Polypeptides and polynucleotides, and uses thereof for treatment of immune related disorders and cancer

ABSTRACT

This invention relates to LY6G6F, VSIG10, TMEM25 and LSR proteins, which are suitable targets for immunotherapy, treatment of cancer, infectious disorders, and/or immune related disorders, and drug development. This invention further relates to soluble LY6G6F, VSIG10, TMEM25 and LSR molecules, extracellular domains of LY6G6F, VSIG10, TMEM25 and LSR and conjugates, which are suitable drugs for immunotherapy, treatment of cancer, infectious disorders, and/or immune related disorders. This invention further relates to antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, specific for LY6G6F, VSIG10, TMEM25 or LSR molecules, which are suitable drugs for immunotherapy, treatment of cancer, infectious disorders, and/or immune related disorders.

FIELD OF THE INVENTION

This invention relates to LY6G6F, VSIG10, TMEM25 and LSR proteins, whichare suitable targets for immunotherapy, treatment of cancer, infectiousdisorders, and/or immune related disorders, and drug development, aswell as soluble molecules and conjugates thereof, and antibodies againstsuch.

BACKGROUND OF THE INVENTION

Naïve T cells must receive two independent signals fromantigen-presenting cells (APC) in order to become productivelyactivated. The first, Signal 1, is antigen-specific and occurs when Tcell antigen receptors encounter the appropriate antigen-MHC complex onthe APC. The fate of the immune response is determined by a second,antigen-independent signal (Signal 2) which is delivered through a Tcell costimulatory molecule that engages its APC-expressed ligand. Thissecond signal could be either stimulatory (positive costimulation) orinhibitory (negative costimulation or coinhibition). In the absence of acostimulatory signal, or in the presence of a coinhibitory signal,T-cell activation is impaired or aborted, which may lead to a state ofantigen-specific unresponsiveness (known as T-cell anergy), or mayresult in T-cell apoptotic death.

Costimulatory molecule pairs usually consist of ligands expressed onAPCs and their cognate receptors expressed on T cells. The prototypeligand/receptor pairs of costimulatory molecules are B7/CD28 andCD40/CD40L. The B7 family consists of structurally related, cell-surfaceprotein ligands, which may provide stimulatory or inhibitory input to animmune response. Members of the B7 family are structurally related, withthe extracellular domain containing at least one variable or constantimmunoglobulin domain.

Both positive and negative costimulatory signals play critical roles inthe regulation of cell-mediated immune responses, and molecules thatmediate these signals have proven to be effective targets forimmunomodulation. Based on this knowledge, several therapeuticapproaches that involve targeting of costimulatory molecules have beendeveloped, and were shown to be useful for prevention and treatment ofcancer by turning on, or preventing the turning off, of immune responsesin cancer patients and for prevention and treatment of autoimmunediseases and inflammatory diseases, as well as rejection of allogenictransplantation, each by turning off uncontrolled immune responses, orby induction of “off signal” by negative costimulation (or coinhibition)in subjects with these pathological conditions.

Manipulation of the signals delivered by B7 ligands has shown potentialin the treatment of autoimmunity, inflammatory diseases, and transplantrejection. Therapeutic strategies include blocking of costimulationusing monoclonal antibodies to the ligand or to the receptor of acostimulatory pair, or using soluble fusion proteins composed of thecostimulatory receptor that may bind and block its appropriate ligand.Another approach is induction of co-inhibition using soluble fusionprotein of an inhibitory ligand. These approaches rely, at leastpartially, on the eventual deletion of auto- or allo-reactive T cells(which are responsible for the pathogenic processes in autoimmunediseases or transplantation, respectively), presumably because in theabsence of costimulation (which induces cell survival genes) T cellsbecome highly susceptible to induction of apoptosis. Thus, novel agentsthat are capable of modulating costimulatory signals, withoutcompromising the immune system's ability to defend against pathogens,are highly advantageous for treatment and prevention of suchpathological conditions.

Costimulatory pathways play an important role in tumor development.Interestingly, tumors have been shown to evade immune destruction byimpeding T cell activation through inhibition of co-stimmulatory factorsin the B7-CD28 and TNF families, as well as by attracting regulatory Tcells, which inhibit anti-tumor T cell responses (see Wang (2006) ImmuneSuppression by Tumor Specific CD4+ Regulatory T cells in Cancer. Semin.Cancer. Biol. 16:73-79; Greenwald, et al. (2005) The B7 FamilyRevisited. Ann. Rev. Immunol. 23:515-48; Watts (2005) TNF/TNFR FamilyMembers in Co-stimulation of T Cell Responses Ann. Rev. Immunol.23:23-68; Sadum, et al. (2007) Immune Signatures of Murine and HumanCancers Reveal Unique Mechanisms of Tumor Escape and New Targets forCancer Immunotherapy. Clin. Cane. Res. 13(13): 4016-4025). Such tumorexpressed co-stimulatory molecules have become attractive cancerbiomarkers and may serve as tumor-associated antigens (TAAs).Furthermore, costimulatory pathways have been identified as immunologiccheckpoints that attenuate T cell dependent immune responses, both atthe level of initiation and effector function within tumor metastases.As engineered cancer vaccines continue to improve, it is becoming clearthat such immunologic checkpoints are a major barrier to the vaccines'ability to induce therapeutic anti-tumor responses. In that regard,costimulatory molecules can serve as adjuvants for active (vaccination)and passive (antibody-mediated) cancer immunotherapy, providingstrategies to thwart immune tolerance and stimulate the immune system.

In addition, such agents could be of use in other types of cancerimmunotherapy, such as adoptive immunotherapy, in which tumor-specific Tcell populations are expanded and directed to attack and kill tumorcells. Agents capable of augmenting such anti-tumor response have greattherapeutic potential and may be of value in the attempt to overcome theobstacles to tumor immunotherapy. Recently, novel agents that modulateseveral costimulatory pathways were indeed introduced to the clinic ascancer immunotherapy.

Emerging data from a wide range of studies on acute and chronicinfections support an important role for negative costimulatoryreceptors also in controlling infection. Memory CD8 T cells generatedafter an acute viral infection are highly functional and constitute animportant component of protective immunity. Modulation of costimulatorypathway has also been proven effective in optimizing antiviral immunityby limiting the memory T cell response to its protective capacities(Teijaro et al., J Immunol. 2009: 182; 5430-5438). This has beendemonstrated in models of influenza infection in which inhibiting CD28costimulation with CTLA4-Ig suppressed primary immune responses in naivemice infected with influenza, but was remarkably curative for memory CD4T cell-mediated secondary responses to influenza leading to improvedclinical outcome and increased survival to influenza challenge.

Chronic infections are often characterized by varying degrees offunctional impairment of virus-specific T-cell responses, and thisdefect is a principal reason for the inability of the host to eliminatethe persisting pathogen. Although functional effector T cells areinitially generated during the early stages of infection, they graduallylose function during the course of the chronic infection as a result ofpersistant exposure to foreign antigen, giving rise to T cellexhaustion. Exhausted T cells express high levels of multipleco-inhibitory receptors such as CTLA-4, PD-1, and LAGS (Crawford et al.,Curr Opin Immunol. 2009; 21:179-186; Kaufmann et al., J Immunol 2009;182:5891-5897, Sharpe et al., Nat Immunol 2007; 8:239-245). PD-1overexpression by exhausted T cells was observed clinically in patientssuffering from chronic viral infections including HIV, HCV and HBV(Crawford et al., Curr Opin Immunol 2009; 21:179-186; Kaufmann et al., JImmunol 2009; 182:5891-5897, Sharpe et al., Nat Immunol 2007;8:239-245). There has been some investigation into this pathway inadditional pathogens, including other viruses, bacteria, and parasites(Hofmeyer et al., J Biomed Biotechnol. Vol 2011, Art. ID 451694, Bhadraet al., Proc Natl Acad Sci. 2011; 108(22):9196-201). For example, thePD-1 pathway was shown to be involved in controlling bacterial infectionusing a sepsis model induced by the standard cecal ligation and puncturemethod. The absence of PD-1 in knockout mice protected fromsepsis-induced death in this model (Huang et al., PNAS 2009: 106;6303-6308).

T cell exhaustion can be reversed by blocking co-inhibitory pathwayssuch as PD-1 or CTLA-4 (Rivas et al., J Immunol. 2009; 183:4284-91;Golden-Mason et al., J Virol. 2009; 83:9122-30; Hofmeyer et al., JBiomed Biotechnol. Vol 2011, Art. ID 451694), thus allowing restorationof anti viral immune function. The therapeutic potential ofco-inhibition blockade for treating viral infection was extensivelystudied by blocking the PD-1/PD-L1 pathway, which was shown to beefficacious in several animal models of infection including acute andchronic simian immunodeficiency virus (SIV) infection in rhesus macaques(Valu et al., Nature 2009; 458:206-210) and in mouse models of chronicviral infection, such as lymphocytic choriomeningitis virus (LCMV)(Barber et al., Nature. 2006; 439:682-7), and Theiler's murineencephalomyelitis virus (TMEV) model in SJL/J mice (Duncan and MillerPLoS One. 2011; 6:e18548). In these models PD-1/PD-L1 blockade improvedanti viral responses and promoted clearance of the persisting viruses.In addition, PD-1/PD-L1 blockade increased the humoral immunitymanifested as elevated production of specific anti-virus antibodies inthe plasma, which in combination with the improved cellular responsesleads to decrease in plasma viral loads and increased survival.

Blocking negative signaling pathways, such as PD-1 and CTLA-4, canrestore the host immune system, enabling it to respond to furtherstimulation. Combining therapeutic vaccination along with the blockadeof inhibitory signals could synergistically enhance functional CD8T-cell responses and improve viral control in chronically infectedindividuals, providing a promising strategy for the treatment of chronicviral infections, such as human immunodeficiency virus, hepatitis Bvirus, and hepatitis C virus (Ha et al, Immunol Rev. 2008 June;223:317-33). The results of a recent study indicate that blocking of thePD-1 pathway improved T cell responses to HBV vaccination in subjectswith HCV infection, and raise the possibility that blocking this pathwaymight improve success rates of immunization in the setting of chronicviral infection (Moorman et al, Vaccine. 2011 Apr. 12; 29(17):3169-76).Antibodies to PD-1 and CTLA-4 are currently in clinical trials inchronic hepatitis C, as promising candidates for combination with bothprophylactic and therapeutic vaccines (Diepolder and Obst, Expert RevVaccines. 2010 March; 9(3):243-7). PD-1 blockade also enhances theeffectiveness of prophylactic vaccination leading to an increase inepitope specific T cells (Finnefrock et al., J Immunol 2009; 182;980-987)

In addition to blockade of co-inhibitory pathways for treatment ofchronic infections, recent studies using viral infection models havehighlighted the importance of positive costimulatory signals duringmemory responses against viruses. Costimulatory molecules such as CD28,4-1BB, and OX40 have also been implicated in the survival, generation,maintenance, and quality of virus-specific memory CD8+T cells. Thedelivery of costimulatory signals can help boost the generation andfunction of virus-specific memory CD8+ T cells. The use of costimulatorymolecules as adjuvants, along with viral antigens in vaccines, mayfacilitate the generation of effective antigen-specific memory CD8+T-cell responses, and may therefore lead to improved vaccines(Duttagupta et al, Crit Rev Immunol. 2009; 29(6):469-86).

A recent study also evaluated the effects of soluble PD-1 (sPD-1) as ablockade of PD-1 and PD-L1 on vaccine-elicited antigen-specific T-cellresponses in mice. Coadministration of sPD-1 with a DNA vaccine or withan adenovirus-based vaccine, increased antigen-specific CD8(+) T-cellresponses, indicating vaccine type-independent adjuvant effect of sPD-1(Song et al, J Immunother. 2011 April; 34(3):297-306). These andadditional results of this study suggest that an immunization strategyusing the soluble extracellular domain (ECD) of a negative costimulatoryprotein as an adjuvant, could be used to increase antigen-specificT-cell immunity elicited by vaccination.

B cells have also long been considered to have a key role in thedevelopment and maintenance of many autoimmune diseases throughproduction of pathogenic autoantibodies, such as systemic lupuserythomatosus (SLE) and Sjogren's disease. However, it is clear that anumber of other B cell functions are also critical in the pathogenesisof organ-specific autoimmune diseases that were previously thought to bemainly T cell mediated, such as rheumatoid arthritis (RA) and type 1diabetes (T1D) (Wong et al 2010, Curr Opin Immunol. 22:723-731).

T cell help to B cells is a pivotal process of adaptive immuneresponses. Follicular helper T (Tfh) cells are a subset of CD4+ T cellsspecialized in B cell help (reviewed by Crotty, Annu. Rev. Immunol. 29:621-663, 2011). Tfh cells express the B cell homing chemokine receptor,CXCR5, which drives Tfh cell migration into B cell follicles withinlymph nodes in a CXCL13-dependent manner Tfh cells first interact withcognate B cells at the T cell-B cell border and subsequently inducegerminal center B cell differentiation and germinal center formationwithin the follicle (Reviewed by Crotty, Annu. Rev. Immunol. 29:621-663, 2011). The requirement of Tfh cells for B cell help and Tcell-dependent antibody responses indicates that this cell type is ofgreat importance for protective immunity against various types ofinfectious agents, as well as for rational vaccine design. Notsurprisingly, dysregulation and aberrant accumulation of Tfh cells hasalso been linked with autoimmune diseases, such as Sjogren's disease andautoimmune arthritis (Yu and Vinuesa, 2010, Cell. Mol. Immunol. 7:198-203).

Tfh cells selectively express a wealth of surface proteins, which areinvolved in their selective localization (such as CXCR5) and in directphysical interactions with B cells to provide B cell help. Among thelatter group are several members of the costimulatory proteins familywhich are highly expressed in Tfh cells, including the inducibleco-stimulatory receptor ICOS, and the negative costimulators (inhibitoryreceptors) PD-1 and BTLA (Crotty, Annu. Rev. Immunol. 29: 621-663,2011), thus this cell subset may be also controlled by modulation ofcostimulatory and coinhibitory pathways, contributing to the effect on Bcell function.

Regulating costimulation using agonists and/or antagonists to variouscostimulatory proteins has been extensively studied as a strategy fortreating autoimmune diseases, graft rejection, allergy and cancer. Thisfield has been clinically pioneered by CTLA4-Ig (Abatacept, Orencia®)which is approved for treatment of RA, mutated CTLA4-Ig (Belatacept,Nulojix®) for prevention of acute kidney transplant rejection and by theanti-CTLA4 antibody (Ipilimumab, Yervoy®), recently approved for thetreatment of melanoma. Other costimulation regulators are currently inadvanced stages of clinical development including anti-PD-1 antibody(MDX-1106) which is in development for treatment of advanced/metastaticclear-cell renal cell carcinoma (RCC) and anti-CD40L Antibody (BG9588,Antova®) for treatment of renal allograft transplantation. Furthermore,such agents are also in clinical development for viral infections, forexample the anti PD-1 Ab, MDX-1106, which is being tested for treatmentof hepatitis C, and the anti-CTLA-4 Ab CP-675,206 (tremelimumab) whichis in a clinical trial in hepatitis C virus-infected patients withhepatocellular carcinoma; the goals of the study are to test its effecton the carcinoma and on the replication of the virus.

BRIEF SUMMARY OF THE INVENTION

According to at least some embodiments, the invention provides noveltherapeutic and diagnostic compositions containing an ectodomain orsoluble or secreted form of the LY6G6F, VSIG10, TMEM25 and/or LSRproteins and/or variants and/or orthologs and/or fragments, and/orconjugate containing same, and/or nucleic acid sequences encoding forsame.

The full length amino acid sequence of the known (wild type) LY6G6Fprotein (lymphocyte antigen 6 complex locus protein G6f, genbankaccession number: NP_001003693, SEQ ID NO:1) is shown in FIG. 1A. Thefull length amino acid sequence of known (wild type) VSIG10 protein(V-set and immunoglobulin domain-containing protein 10, genbankaccession number: NP_061959, SEQ ID NO:3), and the amino acid sequenceof VSIG10 novel variant (SEQ ID NO:5) are shown in FIGS. 1B and 1C,respectively. The amino acid sequence alignment of VSIG10 novel variant(SEQ ID NO:5) and the known (wild type) VSIG10 protein (SEQ ID NO:3) isshown in FIG. 2A. The full length amino acid sequence of known (wildtype) TMEM25 protein (Transmembrane protein 25, Swiss-Prot accessionnumber: Q86YD3, SEQ ID NO:7) is shown in FIG. 1D. The full length aminoacid sequence of known (wild type) LSR protein (lipolysis-stimulatedlipoprotein receptor isoform 2, genbank accession number: NP_991403) isprovided in SEQ ID NO:62. The amino acid sequences of LSR variants SEQID NOs:11, 13, 15, 16, 17 and 18 are shown in FIGS. 1E, IF, 1G, 1H, 1I,and 1J, respectively. The amino acid sequence alignment of the LSRvariants SEQ ID NOs: 11, 13, 15, 16, 17 and 18 with previously known LSRsequences (SEQ ID NOs: 62-67) is demonstrated in FIGS. 2B, 2C, 2D, 2E,2F, 2G, respectively.

According to at least some embodiments, there is provided an isolatedpolypeptide comprising at least 98 amino acids of the soluble ectodomainof a sequence selected from the group consisting of SEQ ID NOs:11, 13,15-18, 67, and 143; at least 62 amino acids of the soluble ectodomain ofa sequence selected from the group consisting of SEQ ID NOs:1 and 58; atleast 36 amino acids of the soluble ectodomain of a sequence selectedfrom the group consisting of SEQ ID NOs:3 and 5; or at least 46 aminoacids of the soluble ectodomain of SEQ ID NO:7, or an isolatedpolypeptide consisting essentially of an amino acid sequence as setforth in SEQ ID NO:5 or variant thereof that possesses at least 95%sequence identity therewith; or variants, or orthologs, or fragmentsthereof.

Optionally the isolated polypeptide comprises only between 98 to 180amino acids of the sequence selected from the group consisting of SEQ IDNOs:11, 13, 15-18, 67, and 143; between 62 to 228 amino acids of thesequence selected from the group consisting of SEQ ID NOs:1 and 58;between 36 and 393 of the sequence selected from the group consisting ofSEQ ID NOs:3 and 5; or between 46 and 216 amino acids of SEQ ID NO:7.

Also optionally, the isolated polypeptide is selected from the groupconsisting of a polypeptide comprising only between 98 to 118, 135 to155, and 160 to 180 amino acids of the sequence selected from the groupconsisting of SEQ ID NOs:11, 13, 15-18, 67, and 143; between 62 to 82,95 to 115, 208 to 228 amino acids of the sequence selected from thegroup consisting of SEQ ID NOs:1 and 58; between 36 to 70, 80 to 100,170 to 200, 265 to 290, 365 to 393 amino acids of the sequence selectedfrom the group consisting of SEQ ID NOs:3 and 5; or between 46 to 66, 84to 104, 196 to 216 amino acids of SEQ ID NO:7.

Also optionally, the isolated polypeptide comprises only about 72, 106,or 218 amino acids of the sequence selected from the group consisting ofSEQ ID NOs:1 and 58; about 108, 145, or 170 amino acids of the sequenceselected from the group consisting of SEQ ID NOs:11, 13, 15-18, 67, and143; about 56, 94, or 206 amino acids of SEQ ID NO:7; or about 46, 49,58, 60, 87, 89, 93, 94, 178, 182, 185, 187, 273, 279, 282, 374 or 383amino acids of SEQ ID NOs:3 and 5.

Also optionally, the isolated polypeptide consists essentially of anamino acid sequence having at least 95% sequence identity with aminoacid sequences set forth in any one of SEQ ID NOs: 12, 2, 4-6, 8, 14,47-50, 10, 15-18, 22, 39, 59-61; 81-102. Optionally and preferably, theisolated polypeptide consists essentially of the amino acid sequence setforth in any one of SEQ ID NOs: 12, 2, 4-6, 8, 14, 47-50, 10, 15-18, 22,39, 59-61; 81-102.

Optionally, the isolated polypeptide blocks or inhibits the interactionof LSR, TMEM25, VSIG10, LY6G6F, or a fragment or variant thereof with acorresponding functional counterpart.

Optionally, the isolated polypeptide replaces or augments theinteraction of LSR, TMEM25, VSIG10, LY6G6F, or a fragment or variantthereof with a corresponding functional counterpart.

Optionally, the isolated ortholog is a mouse polypeptide selected fromSEQ ID NOs: 9 and 19-21.

According to at least some embodiments, the present invention providesisolated polypeptides comprising discrete portions (fragments) of VSIG10proteins, corresponding to:

A. An isolated chimeric polypeptide, comprising a first amino acidsequence being at least 95% homologous toMAAGGSAPEPRVLVCLGALLAGWVAVGLEAVVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGIYTCQEILNVT QWFQVWLQVAcorresponding to amino acids 1-120 of known VSIG10 protein (SEQ IDNO:3), which also corresponds to amino acids 1-120 of VSIG10 variant(SEQ ID NO:5), a second bridging amino acid sequence comprising of N,and a third amino acid sequence being at least 95% homologous toPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLSVKEPLNIGGIVGTIVSLLLLGLAIISGLLLHYSPVFCWKVGNTSRGQNMDDVMVLVDSEEEEEEEEEEEEDAAVGEQEGAREREELPKEIPKQDHIHRVTALVNGNIEQMGNGFQDLQDDSSEEQSDIVQEEDRPV corresponding to amino acids223-540 of known VSIG10 protein (SEQ ID NO:3), which also corresponds toamino acids 122-439 of VSIG10 variant (SEQ ID NO:5), wherein said firstamino acid sequence, second bridging amino acid sequence and third aminoacid sequence are contiguous and in a sequential order.

B. An isolated polypeptide of an edge portion of VSIG10 variant (SEQ IDNO:5), comprising a polypeptide having a length “n”, wherein n is atleast about 10 amino acids in length, optionally at least about 20 aminoacids in length, preferably at least about 30 amino acids in length,more preferably at least about 40 amino acids in length and mostpreferably at least about 50 amino acids in length, wherein at least 3amino acids comprise ANP having a structure as follows (numberingaccording to VSIG10 variant (SEQ ID NO:5)): a sequence starting from anyof amino acid numbers 120-x to 120; and ending at any of amino acidnumbers 122+((n−3)−x), in which x varies from 0 to n−3.

According to at least some embodiments, the subject invention furtherprovides isolated polypeptides comprising a sequence of amino acidresidues corresponding to discrete portions of VSIG10 proteins,corresponding to the new junction and edge portions of VSIG10 variant(SEQ ID NO: 5). The unique sequence of the new junction of VSIG10variant (SEQ ID NO: 5) is demonstrated in protein sequence alignment inFIG. 2A.

According to at least some embodiments, the subject invention providesisolated polypeptides comprising discrete portions (fragments) of LSRproteins, corresponding to:

A. An isolated chimeric polypeptide, comprising a first amino acidsequence being at least 95% homologous toMALLAGGLSRGLGSHPAAAGRDAVVFVWLLLSTWCTAPARAIQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLGRTSGVAELLPGFQAGPIE corresponding to aminoacids 49-258 of known LSR protein (SEQ ID NO:62), which also correspondsto amino acids 1-210 of LSR variant isoform f (SEQ ID NO:18), a secondbridging amino acid sequence comprising of V, and a third amino acidsequence being at least 95% homologous toYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRL KKNLALSRESLVVcorresponding to amino acids 309-649 of known LSR protein (SEQ IDNO:62), which also corresponds to amino acids 212-552 of LSR variantisoform f (SEQ ID NO:18), wherein said first amino acid sequence, secondbridging amino acid and third amino acid sequence are contiguous and ina sequential order.

B. An isolated polypeptide of an edge portion of LSR variant isoform f(SEQ ID NO:18), comprising a polypeptide having a length “n”, wherein nis at least about 10 amino acids in length, optionally at least about 20amino acids in length, preferably at least about 30 amino acids inlength, more preferably at least about 40 amino acids in length and mostpreferably at least about 50 amino acids in length, wherein at least 3amino acids comprise EVY having a structure as follows (numberingaccording to SEQ ID NO:18): a sequence starting from any of amino acidnumbers 210-x to 210; and ending at any of amino acid numbers212+((n−3)−x), in which x varies from 0 to n−3.

C. An isolated chimeric polypeptide comprising a first amino acidsequence being at least 95% homologous toMALLAGGLSRGLGSHPAAAGRDAVVFVWLLLSTWCTAPARAIQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVL corresponding to amino acids 49-239 ofknown LSR protein (SEQ ID NO:66), which also corresponds to amino acids1-191 of LSR variant isoform f (SEQ ID NO:18), a second amino acidsequence being at least 80%, preferably at least 85%, more preferably atleast 90% and most preferably at least 95% homologous to a polypeptidehaving the sequence GRTSGVAELLPGFQAGPIE corresponding to amino acids192-218 of LSR variant isoform f (SEQ ID NO:18), and a third amino acidsequence being at least 95% homologous toVYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV corresponding to amino acids 240-581 of known LSRprotein SEQ ID NO:66, which also corresponds to amino acids 211-552 ofLSR variant isoform f (SEQ ID NO:18), wherein said first amino acidsequence, second amino acid sequence and third amino acid sequence arecontiguous and in a sequential order.

D. An isolated polypeptide of an edge portion of LSR variant isoform f(SEQ ID NO:18), comprising an amino acid sequence being at least about80%, preferably at least about 85%, more preferably at least about 90%and most preferably at least about 95% homologous to the sequenceGRTSGVAELLPGFQAGPIE of LSR variant isoform f (SEQ ID NO:18).

According to at least some embodiments, the subject invention furtherprovides isolated polypeptides comprising a sequence of amino acidresidues corresponding to discrete portions of LSR, corresponding to thenew junction and edge portions of LSR variant LSR isoform-f (SEQ ID NO:18). The unique sequences of the new junction of the LSR isoform-f (SEQID NO: 18) is demonstrated in protein sequence alignment in FIG. 2G.

According to at least some embodiments, the subject invention providespolypeptides comprising a sequence of amino acid residues correspondingto discrete portions of LY6G6F, VSIG10, TMEM25 and/or LSR proteins,including different portions of the extracellular domain correspondingto residues 17-234 of LY6G6F (SEQ ID NO:1), corresponding to amino acidsequence depicted in SEQ ID NO:2; residues 31-413 of VSIG10 (SEQ IDNO:3), corresponding to amino acid sequence depicted in SEQ ID NO:4;residues 31-312 of VSIG10 (SEQ ID NO:5), corresponding to amino acidsequence depicted in SEQ ID NO:6; residues 27-232 of TMEM25 (SEQ IDNO:7), corresponding to amino acid sequence depicted in SEQ ID NO:8;residues 42-211 of LSR (SEQ ID NO:11, and/or SEQ ID NO:143),corresponding to amino acid sequence depicted in SEQ ID NO:12; residues42-192 of LSR (SEQ ID NO:13), corresponding to amino acid sequencedepicted in SEQ ID NO:14, residues 42-533 of LSR (SEQ ID NO:15),corresponding to amino acid sequence depicted in SEQ ID NO:47, residues42-532 of LSR (SEQ ID NO:16), corresponding to amino acid sequencedepicted in SEQ ID NO:48, residues 42-493 of LSR (SEQ ID NO:17),corresponding to amino acid sequence depicted in SEQ ID NO:49, residues42-552 of LSR (SEQ ID NO:18), corresponding to amino acid sequencedepicted in SEQ ID NO:50, and/or fragments and/or variants thereofpossessing at least 85%, 90%, 95, 96, 97, 98 or 99% sequence homologytherewith. According to still further embodiments, the LY6G6F ECDfragments are selected from any one of SEQ ID NOs 81, 96, and variantsthereof, as described herein. According to still further embodiments,the VSIG10 ECD fragments are selected from any one of SEQ ID NOs 82-93,97-100, and variants thereof, as described herein. According to stillfurther embodiments, the LSR ECD fragments are selected from any one ofSEQ ID NOs 95, 102, and variants thereof, as described herein. Accordingto still further embodiments, the TMEM25 ECD fragments are selected fromany one of SEQ ID NOs 94, 101, and variants thereof, as describedherein. According to still further embodiments, the discrete portions ofLY6G6F, VSIG10, TMEM25 and/or LSR proteins may or may not include asignal (leader) peptide (SP) sequence (FIG. 1). According to at leastsome embodiments of the invention, there are provided examples of theECD portions including SP sequences of LY6G6F, VSIG10, TMEM25 and/or LSRproteins. An example of ECD portion including SP sequence of LY6G6Fprotein (SEQ ID NO:1) is amino acid sequence set forth in SEQ ID NO:59.An example of ECD portion including SP sequence of VSIG10 protein (SEQID NO:3) is amino acid sequence set forth in SEQ ID NO:60. An example ofECD portion including SP sequence of VSIG10 protein (SEQ ID NO:5) isamino acid sequence set forth in SEQ ID NO:61. An example of ECD portionincluding SP sequence of TMEM25 protein (SEQ ID NO:7) is amino acidsequence set forth in SEQ ID NO: 39. An example of ECD portion includingSP sequence of LSR protein (SEQ ID NO:11) is amino acid sequence setforth in SEQ ID NO:10. An example of ECD portion including SP sequenceof LSR protein (SEQ ID NO:14) is amino acid sequence set forth in SEQ IDNO:22.

According to further embodiments, the invention provides polypeptidescomprising a sequence of amino acid residues corresponding to solubleLSR proteins depicted in SEQ ID NO: 18, including different portionsthereof or variants thereof possessing at least 85%, 90%, 95, 96, 97, 98or 99% sequence homology therewith. According to further embodiments,the invention provides polypeptides comprising a sequence of amino acidresidues corresponding to soluble LSR proteins depicted in any one ofSEQ ID NOs:15-16, including different portions thereof or variantsthereof possessing at least 95, 96, 97, 98 or 99% sequence homologytherewith. According to further embodiments, the invention providespolypeptides comprising a sequence of amino acid residues correspondingto soluble LSR proteins depicted in any one of SEQ ID NOs:15-18.According to still further embodiments, the soluble LSR proteinsdepicted in any one of SEQ ID NOs:15-18 may or may not include a signal(leader) peptide sequence (FIGS. 1G, G, I and J).

According to still further embodiments, the invention providespolypeptides comprising a sequence of amino acid residues correspondingto extracellular domains of orthologs of TMEM25, LY6G6F, VSIG10, LSRvariant 1 and/or LSR variant 2 proteins, particularly mouse orthologs(SEQ ID NOs: 28, 29, 30, 31 and/or 32, respectively), including but notlimited to mouse orthologs extracellular domains corresponding to aminoacid sequence depicted in SEQ ID NOs: 9, 19-21, or portions or variantsthereof possessing at least 85%, 90%, 95, 96, 97, 98 or 99% sequencehomology therewith.

According to still further embodiments, the invention providespolypeptides comprising an amino acid sequence corresponding to any oneof novel variants of VSIG10 (SEQ ID NO: 5), and LSR (SEQ ID NOs: 11, 13,15, 16 and 18).

According to at least some embodiments, the present invention provides afusion protein comprising any of the above polypeptides joined to aheterologous sequence. Optionally, the heterologous sequence comprisesat least a portion of an immunoglobulin molecule. Optionally andpreferably, the immunoglobulin molecule portion is an immunoglobulinheavy chain constant region Fc fragment. Optionally and more preferably,the immunoglobulin heavy chain constant region is derived from animmunoglobulin isotype selected from the group consisting of an IgG1,IgG2, IgG3, IgG4, IgM, IgE, IgA and IgD. Optionally and most preferably,the fusion protein has the amino acid sequence set forth in any one ofSEQ ID NOs: 71-80, 172-181 or set forth in any one of SEQ ID NOs:23-26and also optionally modulates immune cell response in vitro or in vivo.

According to at least some embodiments, the subject invention providesisolated nucleic acid sequences encoding any one of the foregoing novelvariants of TMEM25, VSIG10, and/or LSR and/or any one of the foregoingLY6G6F, VSIG10, TMEM25 and/or LSR extracellular domain polypeptides orfragments or homologs or orthologs thereof.

According to at least some embodiments, there is provided an isolatednucleic acid sequence selected from the group consisting of SEQ ID NOs:33-37, 40-46, 132, 155, 182-198, or variant thereof that possesses atleast 95% sequence identity therewith, or a degenerative variantthereof.

According to at least some embodiments, the subject invention providesan isolated polynucleotide encoding a polypeptide comprising any one ofthe amino acid sequences, as set forth in SEQ ID NOs: 2, 4, 5, 6, 8-16,18-22, 39, 47-50, 59-61, 143, or a fragment or variant thereof thatpossesses at least 85, 90, 95, 96, 97, 98 or 99% sequence identitytherewith, or a degenerative variant thereof.

According to at least some embodiments, the subject invention providesan isolated polynucleotide comprising a nucleic acid as set forth in anyone of SEQ ID NO:33-37, 40-46, 132, 145, 155, 182-188, or a sequencehomologous thereto or degenerative variants thereof. According toanother embodiment, the isolated polynucleotide is at least 85, 90, 95,96, 97, 98 or 99% homologous to a nucleic acid sequence as set forth inany one of SEQ ID NOs: 33-37, 40-46, 145.

According to at least some embodiments, there is provided an expressionvector or a virus, containing at least one isolated nucleic acidsequence as described herein. According to at least some embodiments,there is provided a recombinant cell comprising an expression vector ora virus containing an isolated nucleic acid sequence as describedherein, wherein the cell constitutively or inducibly expresses thepolypeptide encoded by the DNA segment. According to at least someembodiments, there is provided a method of producing a LSR, TMEM25,VSIG10, LY6G6F soluble ectodomain polypeptide, or fragment or fusionprotein thereof, comprising culturing the recombinant cell as describedherein, under conditions whereby the cell expresses the polypeptideencoded by the DNA segment or nucleic acid and recovering saidpolypeptide.

According to at least some embodiments of the present invention, thereis provided a pharmaceutical composition comprising an isolated aminoacid sequence of ectodomain or soluble or secreted forms of any one ofLY6G6F, VSIG10, TMEM25, LSR proteins or variants or orthologs orfragments or conjugates containing same.

According to at least some embodiments, the invention provides anisolated or purified amino acid sequence of soluble and/or extracellulardomain of LY6G6F, VSIG10, TMEM25 and/or LSR protein or nucleic acidsequence encoding same, which optionally may be directly or indirectlyattached to a non-LY6G6F, VSIG10, TMEM25 and/or LSR protein or nucleicacid sequence, such as a soluble immunoglobulin domain or fragment.

According to at least some embodiments, the invention provides vectorssuch as plasmids and recombinant viral vectors and host cells containingthat express secreted or soluble form and/or the ECD of the LY6G6F,VSIG10, TMEM25 and/or LSR protein or fragments or variants or orthologsthereof or polypeptide conjugates containing any of the foregoing.

According to at least some embodiments the invention provides a use ofthese vectors such as plasmids and recombinant viral vectors and hostcells containing that express any one of LY6G6F, VSIG10, TMEM25 and/orLSR, secreted and/or soluble form and/or the ECD and/or fragmentsthereof and/or variants, and/or orthologs thereof and/or polypeptideconjugates containing any of the foregoing to produce any one of saidLY6G6F, VSIG10, TMEM25 and/or LSR proteins.

According to at least some embodiments, the invention providespharmaceutical or diagnostic compositions containing any of theforegoing.

According to at least some embodiments, the invention provides a use ofany one of the compounds containing at least one of LY6G6F, VSIG10,TMEM25 and/or LSR ectodomains, soluble or secreted form or fragments ororthologs or variants thereof, or conjugates, or nucleic acid sequenceencoding same, or pharmaceutical composition comprising same, astherapeutics for treatment or prevention of cancer as recited herein,infectious disorder as recited herein, and/or immune related disorder,including but not limited to autoimmune diseases as recited herein,transplant rejection and graft versus host disease and/or for blockingor promoting immune costimulation mediated by any one of the LY6G6F,VSIG10, TMEM25 and/or LSR polypeptides, immune related diseases asrecited herein and/or for immunotherapy (promoting or inhibiting immunecostimulation). According to at least some embodiments, the autoimmunedisease includes any autoimmune disease, and optionally and preferablyincludes but is not limited to any of the types and subtypes of any ofmultiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis,systemic lupus erythematosus, inflammatory bowel disease, uveitis, orSjogren's syndrome.

According to at least some embodiments, the invention provides a use ofany one of the compounds containing at least one of LY6G6F, VSIG10,TMEM25 and/or LSR ectodomains, soluble or secreted form or fragments ororthologs or variants thereof, or conjugates, or nucleic acid sequenceencoding same, or pharmaceutical composition comprising same, foradministration as an anti-cancer vaccine, as an adjuvant for anti cancervaccine, and/or for adoptive immunotherapy, and/or for immunotherapy ofcancer as recited herein.

According to at least some embodiments, the invention provides a use ofany of the LY6G6F, VSIG10, TMEM25 and/or LSR proteins, and/or nucleicacid sequences as targets for development of drugs which specificallybind to any one of the LY6G6F, VSIG10, TMEM25 and/or LSR proteins and/ordrugs which agonize or antagonize the binding of other moieties to theLY6G6F, VSIG10, TMEM25 and/or LSR proteins.

According to at least some embodiments, the present invention providesdrugs which modulate (agonize or antagonize) at least one of the LY6G6F,VSIG10, TMEM25 and/or LSR related biological activity. Such drugsinclude by way of example antibodies, small molecules, peptides,ribozymes, aptamers, antisense molecules, siRNA's and the like. Thesemolecules may directly bind or modulate an activity elicited by the anyone of the LY6G6F, VSIG10, TMEM25 and/or LSR proteins or the LY6G6F,VSIG10, TMEM25 and/or LSR DNA or portions or variants thereof or mayindirectly modulate any one of the LY6G6F, VSIG10, TMEM25 and/or LSRassociated activity or binding of molecules to any one of the LY6G6F,VSIG10, TMEM25 and/or LSR and portions and variants thereof such as bymodulating the binding of any one of LY6G6F, VSIG10, TMEM25 and/or LSRto its counterreceptor or endogenous ligand.

According to at least some embodiments, the invention provides novelmonoclonal or polyclonal antibodies and antigen binding fragments andconjugates containing same, and/or alternative scaffolds, thatspecifically bind any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteinsas described herein or polypeptides having at least 95% homologythereto. Optionally such antibodies bind to proteins selected from thegroup consisting of any one of SEQ ID NOs: 1-8, 10-18, 22, 39, 47-50,59-61, 9, 19-21, and/or the amino acid sequences corresponding to theunique edges of any one of SEQ ID NOs: 5 and 18, particularly whereinthese antibodies, antigen binding fragments and conjugates containingsame, and/or alternative scaffolds, are adapted to be used astherapeutic and/or diagnostic agents (both in vitro and in vivodiagnostic methods), particularly for treatment and/or diagnosis ofinfectious disorder as recited herein, and/or immune related disorder,including but not limited to autoimmune diseases as recited herein,immune related diseases as recited herein, transplant rejection andgraft versus host disease, as well as cancers and malignancies asrecited herein.

According to at least some embodiments, there are provided antibodies inwhich the antigen binding site comprises a conformational or linearepitope, and wherein the antigen binding site contains about 3-7contiguous or non-contiguous amino acids. Optionally, the antibody is afully human antibody, chimeric antibody, humanized or primatizedantibody.

Also optionally, the antibody is selected from the group consisting ofFab, Fab′, F(ab′)2, F(ab′), F(ab), Fv or scFv fragment and minimalrecognition unit.

Also optionally, the antibody is coupled to a moiety selected from adrug, a radionuclide, a fluorophore, an enzyme, a toxin, a therapeuticagent, or a chemotherapeutic agent; and wherein the detectable marker isa radioisotope, a metal chelator, an enzyme, a fluorescent compound, abioluminescent compound or a chemiluminescent compound.

Also optionally the antibody blocks or inhibits the interaction of anyone of LSR, TMEM25, VSIG10, LY6G6F polypeptides, or a fragment orvariant thereof with a counterpart.

Also optionally the antibody replaces or augments the interaction ofLSR, TMEM25, VSIG10, LY6G6F polypeptides, or a fragment or variantthereof with a counterpart.

Also optionally the antibody elicits apoptosis or lysis of cancer cellsthat express any one of LSR, TMEM25, VSIG10, LY6G6F protein.

Also optionally the apoptosis or lysis involves CDC or ADCC activity ofthe antibody, wherein CDC (complement dependent cytotoxicity) or ADCC(antibody dependent cellular cytotoxicity) activities are used to targetthe immune cells.

According to at least some embodiments, the invention providesantibodies and antigen binding fragments and conjugates containing same,and/or alternative scaffolds, against discrete portions of the LY6G6Fprotein including different portions of the extracellular domaincorresponding to residues 17-234 of LY6G6F (SEQ ID NO:1), set forth inSEQ ID NO: 2, and/or corresponding to amino acid sequences set forth inany one of SEQ ID NOs: 81, 96. According to further embodiments theinvention provides antibodies antigen binding fragments and conjugatescontaining same, and/or alternative scaffolds, against discrete portionsof the mouse LY6G6F protein (SEQ ID NO: 29), including differentportions of the extracellular domain corresponding to SEQ ID NO:20.

According to at least some embodiments, the invention providesantibodies and antigen binding fragments and conjugates containing same,and/or alternative scaffolds, against discrete portions of the VSIG10protein including different portions of the extracellular domaincorresponding to amino acid residues 31-413 of VSIG10 (SEQ ID NO:3),depicted in SEQ ID NO:4; amino acid residues 31-312 of VSIG10 (SEQ IDNO:5), depicted in SEQ ID NO:6, and/or corresponding to amino acidsequences set forth in any one of SEQ ID NOs:82-93, 97-100. According tofurther embodiments the invention provides antibodies antigen bindingfragments and conjugates containing same, and/or alternative scaffolds,against discrete portions of the mouse VSIG10 protein (SEQ ID NO: 30),including different portions of the extracellular domain correspondingto SEQ ID NO:19. According to at least some embodiments, the inventionprovides antibodies, antigen binding fragments and conjugates containingsame, and/or alternative scaffolds, against discrete portions of theVSIG10 protein including the edge portion of VSIG10 variant (SEQ IDNO:5), as described herein.

According to at least some embodiments, the invention providesantibodies, antigen binding fragments and conjugates containing same,and/or alternative scaffolds, against discrete portions of the TMEM25proteins including different portions of the extracellular domaincorresponding to amino acid residues 27-232 of TMEM25 (SEQ ID NO:7),depicted in SEQ ID NO:8, and/or corresponding to amino acid sequencesset forth in any one of SEQ ID NOs: 94, 101. According to furtherembodiments the invention provides antibodies and antigen bindingfragments and conjugates containing same, and/or alternative scaffolds,against discrete portions of the mouse TMEM25 protein (SEQ ID NO: 28),including different portions of the extracellular domain, set forth inSEQ ID NO:9.

According to at least some embodiments, the invention providesantibodies and antigen binding fragments and conjugates containing same,and/or alternative scaffolds, against discrete portions of the LSRproteins including different portions of the extracellular domaincorresponding to amino acid residues 42-211 of LSR (SEQ ID NO:11),depicted in SEQ ID NO:12; amino acid residues 42-192 of LSR (SEQ IDNO:13), depicted in SEQ ID NO:14, amino acid residues 42-533 of LSR (SEQID NO:15), depicted in SEQ ID NO:47, amino acid residues 42-532 of LSR(SEQ ID NO:16), depicted in SEQ ID NO:48, amino acid residues 42-493 ofLSR (SEQ ID NO:17), depicted in SEQ ID NO:49, amino acid residues 42-552of LSR (SEQ ID NO:18), depicted in SEQ ID NO:50, and/or corresponding toamino acid sequences set forth in any one of SEQ ID NOs:95, 102.According to further embodiments the invention provides antibodies andantigen binding fragments and conjugates containing same, and/oralternative scaffolds, against discrete portions of the mouse LY6G6Fproteins (SEQ ID NOs: 31-32), including different portions of theextracellular domain corresponding to SEQ ID NO:21.

According to at least some embodiments, the invention providesantibodies and antigen binding fragments and conjugates containing same,and/or alternative scaffolds, against discrete portions of the LSRproteins including the unique edge portion of LSR variant isoform-f (SEQID NO:18), as described herein.

According to at least some embodiments, the invention providesantibodies and antigen binding fragments and conjugates containing same,and/or alternative scaffolds, against discrete portions of the solubleLSR proteins including different portions of the LSR proteins depictedin any one of SEQ ID NOs:15-18, 47-50.

According to at least some embodiments the invention relates to proteinscaffolds with specificities and affinities in a range similar tospecific antibodies. According to at least some embodiments the presentinvention relates to an antigen-binding construct comprising a proteinscaffold which is linked to one or more epitope-binding domains. Suchengineered protein scaffolds are usually obtained by designing a randomlibrary with mutagenesis focused at a loop region or at an otherwisepermissible surface area and by selection of variants against a giventarget via phage display or related techniques. According to at leastsome embodiments the invention relates to alternative scaffoldsincluding, but not limited to, anticalins, DARPins, Armadillo repeatproteins, protein A, lipocalins, fibronectin domain, ankyrin consensusrepeat domain, thioredoxin, chemically constrained peptides and thelike. According to at least some embodiments the invention relates toalternative scaffolds that are used as therapeutic agents for treatmentof cancer as recited herein, immune related diseases as recited herein,autoimmune disease as recited herein and infectious diseases, as well asfor in vivo diagnostics.

According to at least some embodiments of the present invention, thereis provided a pharmaceutical composition comprising an isolatedpolypeptide as described herein, or a fusion protein as describedherein; a nucleotide sequence as described herein; an expression vectoras described herein; a host cell as described herein, or an antibody asdescribed herein, and further comprising a pharmaceutically acceptablediluent or carrier.

According to at least some embodiments, there is provided use of any ofany one of an isolated polypeptide as described herein, or a fusionprotein as described herein; a nucleotide sequence as described herein;an expression vector as described herein; a host cell as describedherein, or an antibody as described herein or a pharmaceuticalcomposition as described herein, wherein administration of such to thesubject inhibits or reduces activation of T cells.

According to at least some embodiments, there is provided use of any ofany one of an isolated polypeptide as described herein, or a fusionprotein as described herein; a nucleotide sequence as described herein;an expression vector as described herein; a host cell as describedherein, or an antibody as described herein or a pharmaceuticalcomposition as described herein, for treatment of cancer.

According to at least some embodiments, there is provided use of anisolated polypeptide as described herein, or a fusion protein asdescribed herein; a nucleotide sequence as described herein; anexpression vector as described herein; a host cell as described herein,or an antibody as described herein or a pharmaceutical composition asdescribed herein, for treatment of infectious disorder.

According to at least some embodiments, there is provided a method ofperforming one or more of the following in a subject:

a. upregulating cytokines;

b. inducing expansion of T cells;

c. promoting antigenic specific T cell immunity;

d. promoting CD4+ and/or CD8+ T cell activation;

comprising administering any of an isolated polypeptide as describedherein, or a fusion protein as described herein; a nucleotide sequenceas described herein; an expression vector as described herein; a hostcell as described herein, or an antibody as described herein or apharmaceutical composition as described hereinto the subject.

According to at least some embodiments, there is provided a method fortreating or preventing immune system related condition comprisingadministering to a subject in need thereof an effective amount of any ofan isolated polypeptide as described herein, or a fusion protein asdescribed herein; a nucleotide sequence as described herein; anexpression vector as described herein; a host cell as described herein,or an antibody as described herein or a pharmaceutical composition.

Optionally, the immune system related condition comprises an immunerelated condition, autoimmune diseases as recited herein, transplantrejection and graft versus host disease and/or for blocking or promotingimmune costimulation mediated by any one of the LSR, TMEM25, VSIG10,and/or LY6G6F polypeptides, immune related diseases as recited hereinand/or for immunotherapy (promoting or inhibiting immune costimulation).

Optionally the treatment is combined with another moiety useful fortreating immune related condition.

Optionally the moiety is selected from the group consisting ofimmunosuppressants such as corticosteroids, cyclosporin,cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin,tacrolimus, biological agents such as TNF-alpha blockers or antagonists,or any other biological agent targeting any inflammatory cytokine,nonsteroidal antiinflammatory drugs/Cox-2 inhibitors,hydroxychloroquine, sulphasalazopryine, gold salts, etanercept,infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab,cytoxan, interferon beta-1a, interferon beta-1b, glatiramer acetate,mitoxantrone hydrochloride, anakinra and/or other biologics and/orintravenous immunoglobulin (IVIG), interferons such as IFN-beta-1a(REBIF®. and AVONEX®) and IFN-beta-1b (BETASERON®); glatiramer acetate(COPAXONE®), a polypeptide; natalizumab (TYSABRI®), mitoxantrone(NOVANTRONE®), a cytotoxic agent, a calcineurin inhibitor, e.g.cyclosporin A or FK506; an immunosuppressive macrolide, e.g. rapamycineor a derivative thereof; e.g. 40-O-(2-hydroxy)ethyl-rapamycin, alymphocyte homing agent, e.g. FTY720 or an analog thereof,corticosteroids; cyclophosphamide; azathioprene; methotrexate;leflunomide or an analog thereof; mizoribine; mycophenolic acid;mycophenolate mofetil; 15-deoxyspergualine or an analog thereof;immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies toleukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD 11a/CD18, CD7, CD25,CD 27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB ortheir ligands; or other immunomodulatory compounds, e.g. CTLA4-Ig(abatacept, ORENCIA®), CD28-Ig, B7-H4-Ig, or other costimulatory agents,or adhesion molecule inhibitors, e.g. mAbs or low molecular weightinhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4antagonists, or another immunomodulatory agent.

Optionally the immune condition is selected from autoimmune disease,transplant rejection, or graft versus host disease.

Optionally the autoimmune disease is selected from a group consisting ofmultiple sclerosis, including relapsing-remitting multiple sclerosis,primary progressive multiple sclerosis, and secondary progressivemultiple sclerosis; psoriasis; rheumatoid arthritis; psoriaticarthritis, systemic lupus erythematosus (SLE); ulcerative colitis;Crohn's disease; benign lymphocytic angiitis, thrombocytopenic purpura,idiopathic thrombocytopenia, idiopathic autoimmune hemolytic anemia,pure red cell aplasia, Sjogren's syndrome, rheumatic disease, connectivetissue disease, inflammatory rheumatism, degenerative rheumatism,extra-articular rheumatism, juvenile rheumatoid arthritis, arthritisuratica, muscular rheumatism, chronic polyarthritis, cryoglobulinemicvasculitis, ANCA-associated vasculitis, antiphospholipid syndrome,myasthenia gravis, autoimmune haemolytic anaemia, Guillian-Barresyndrome, chronic immune polyneuropathy, autoimmune thyroiditis, insulindependent diabetes mellitus, type I diabetes, Addison's disease,membranous glomerulonephropathy, Goodpasture's disease, autoimmunegastritis, autoimmune atrophic gastritis, pernicious anaemia, pemphigus,pemphigus vulgarus, cirrhosis, primary biliary cirrhosis,dermatomyositis, polymyositis, fibromyositis, myogelosis, celiacdisease, immunoglobulin A nephropathy, Henoch-Schonlein purpura, Evanssyndrome, atopic dermatitis, psoriasis, psoriasis arthropathica, Graves'disease, Graves' ophthalmopathy, scleroderma, systemic scleroderma,progressive systemic scleroderma, asthma, allergy, primary biliarycirrhosis, Hashimoto's thyroiditis, primary myxedema, sympatheticophthalmia, autoimmune uveitis, hepatitis, chronic action hepatitis,collagen diseases, ankylosing spondylitis, periarthritishumeroscapularis, panarteritis nodosa, chondrocalcinosis, Wegener'sgranulomatosis, microscopic polyangiitis, chronic urticaria, bullousskin disorders, pemphigoid, atopic eczema, Devic's disease, childhoodautoimmune hemolytic anemia, Refractory or chronic AutoimmuneCytopenias, Prevention of development of Autoimmune Anti-Factor VIIIAntibodies in Acquired Hemophilia A, Cold Agglutinin Disease,Neuromyelitis Optica, Stiff Person Syndrome, gingivitis, periodontitis,pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis,and inflammatory skin disorders, selected from the group consisting ofpsoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne,normocomplementemic urticarial vasculitis, pericarditis, myositis,anti-synthetase syndrome, scleritis, macrophage activation syndrome,Bechet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult andjuvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome,familial cold-induced auto-inflammatory syndrome, neonatal onsetmultisystemic inflammatory disease, familial Mediterranean fever,chronic infantile neurologic, cutaneous and articular syndrome, systemicjuvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler'ssyndrome, autoimmune retinopathy, age-related macular degeneration,atherosclerosis, chronic prostatitis and TNF receptor-associatedperiodic syndrome (TRAPS).

Optionally the autoimmune disease is selected from the group consistingof any of the types and subtypes of any of multiple sclerosis,rheumatoid arthritis, type I diabetes, psoriasis, systemic lupuserythematosus, inflammatory bowel disease, uveitis, and Sjogren'ssyndrome.

According to at least some embodiments there is provided a method fortreating or preventing an infectious disease comprising administering toa subject in need thereof an effective amount of any of an isolatedpolypeptide as described herein, or a fusion protein as describedherein; a nucleotide sequence as described herein; an expression vectoras described herein; a host cell as described herein, or an antibody asdescribed herein or a pharmaceutical composition.

Optionally the infectious disease is selected from the disease caused bybacterial infection, viral infection, fungal infection and/or otherparasite infection.

Optionally the infectious disease is selected from hepatitis B,hepatitis C, infectious mononucleosis, EBV, cytomegalovirus, AIDS,HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.

According to at least some embodiments, there is provided a method fortreating or preventing cancer comprising administering to a subject inneed thereof an effective amount of any of an isolated polypeptide asdescribed herein, or a fusion protein as described herein; a nucleotidesequence as described herein; an expression vector as described herein;a host cell as described herein, or an antibody as described herein or apharmaceutical composition.

Optionally the treatment is combined with another moiety or therapyuseful for treating cancer.

Optionally the therapy is radiation therapy, antibody therapy,chemotherapy, photodynamic therapy, adoptive T cell therapy, Tregdepletion, surgery or in combination therapy with conventional drugs.

Optionally the moiety is selected from the group consisting ofimmunosuppressants, cytotoxic drugs, tumor vaccines, antibodies (e.g.bevacizumab, erbitux), peptides, pepti-bodies, small molecules,chemotherapeutic agents such as cytotoxic and cytostatic agents (e.g.paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine,temozolomide, irinotecan, 5FU, carboplatin), immunological modifierssuch as interferons and interleukins, immunostimulatory antibodies,growth hormones or other cytokines, folic acid, vitamins, minerals,aromatase inhibitors, RNAi, Histone Deacetylase Inhibitors, andproteasome inhibitors.

Optionally the cancer is selected from a group consisting of breastcancer, cervical cancer, ovary cancer, endometrial cancer, melanoma,bladder cancer, lung cancer, pancreatic cancer, colon cancer, prostatecancer, leukemia, acute lymphocytic leukemia, chronic lymphocyticleukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma,Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myeloid leukemia, acutemyelogenous leukemia (AML), chronic myelogenous leukemia, thyroidcancer, thyroid follicular cancer, myelodysplastic syndrome (MDS),fibrosarcomas and rhabdomyosarcomas, melanoma, uveal melanoma,teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor ofthe skin, keratoacanthomas, renal cancer, anaplastic large-celllymphoma, esophageal squamous cells carcinoma, hepatocellular carcinoma,follicular dendritic cell carcinoma, intestinal cancer, muscle-invasivecancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer,bladder cancer, head and neck cancer, stomach cancer, liver cancer, bonecancer, brain cancer, cancer of the retina, biliary cancer, small bowelcancer, salivary gland cancer, cancer of uterus, cancer of testicles,cancer of connective tissue, prostatic hypertrophy, myelodysplasia,Waldenstrom's macroglobinaemia, nasopharyngeal, neuroendocrine cancer,myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma,carcinoid, oesophagogastric, fallopian tube cancer, peritoneal cancer,papillary serous mullerian cancer, malignant ascites, gastrointestinalstromal tumor (GIST), Li-Fraumeni syndrome and Von Hippel-Lindausyndrome (VHL), and wherein the cancer is non-metastatic, invasive ormetastatic.

Optionally the cancer is any of melanoma, cancer of liver, renal, brain,breast, colon, lung, ovary, pancreas, prostate, stomach, multiplemyeloma, Hodgkin's lymphoma, non Hodgkin's lymphoma, acute and chroniclymphoblastic leukemia and acute and chronic myeloid leukemia.

According to at least some embodiments, there is provided a method forpotentiating a secondary immune response to an antigen in a patient,which method comprises administering effective amount of any of anisolated polypeptide as described herein, or a fusion protein asdescribed herein; a nucleotide sequence as described herein; anexpression vector as described herein; a host cell as described herein,or an antibody as described herein or a pharmaceutical composition.

Optionally the antigen is a cancer antigen, a viral antigen or abacterial antigen, and the patient has received treatment with ananticancer vaccine or a viral vaccine.

A method of immunotherapy in a patient, comprising:

in vivo or ex vivo tolerance induction, comprising administeringeffective amount of any of an isolated polypeptide as described herein,or a fusion protein as described herein; a nucleotide sequence asdescribed herein; an expression vector as described herein; a host cellas described herein, or an antibody as described herein or apharmaceutical composition, to a patient or to leukocytes isolated fromthe patient, in order to induce differentiation of tolerogenicregulatory cells;

ex-vivo enrichment and expansion of said cells;

reinfusion of the tolerogenic regulatory cells to said patient.

A method of using at least one of: any of an isolated polypeptide asdescribed herein, or a fusion protein as described herein; a nucleotidesequence as described herein; an expression vector as described herein;a host cell as described herein, or an antibody as described herein or apharmaceutical composition; as a cancer vaccine adjuvant, comprisingadministration to a patient an immunogenic amount of a tumor associatedantigen preparation of interest; and a cancer vaccine adjuvant in aformulation suitable for immunization, wherein the immune responseagainst the tumor associated antigen in the presence of the cancervaccine adjuvant is stronger than in the absence of the cancer vaccineadjuvant.

According to at least some embodiments there is provided a method forcombining therapeutic vaccination with an antigen along withadministration of any of an isolated polypeptide as described herein, ora fusion protein as described herein; a nucleotide sequence as describedherein; an expression vector as described herein; a host cell asdescribed herein, or an antibody as described herein or a pharmaceuticalcomposition, for treatment of infection.

According to at least some embodiments, there is provided a method forcombining any of an isolated polypeptide as described herein, or afusion protein as described herein; a nucleotide sequence as describedherein; an expression vector as described herein; a host cell asdescribed herein, or an antibody as described herein or a pharmaceuticalcomposition, an adjuvant, and an antigen in a vaccine, in order toincrease the immune response.

Optionally the antigen is a viral antigen, bacterial antigen, fungalantigen, parasite antigen, and/or other pathogen's antigen.

According to at least some embodiments, any one of the foregoingtherapeutic agents according to at least some embodiments of the presentinvention, including antibodies and antigen binding fragments andconjugates containing same, and/or alternative scaffolds, against anyone of LY6G6F, VSIG10, TMEM25 and/or LSR proteins; LY6G6F, VSIG10,TMEM25 and/or LSR secreted or soluble form or ECD and/or variants,and/or orthologs, and/or conjugates thereof, can be used for adoptiveimmunotherapy. Immune tolerance or immunological tolerance is theprocess by which the immune system does not attack an antigen. It can beeither ‘natural’ or ‘self tolerance’, where the body does not mount animmune response to self antigens, or ‘induced tolerance’, wheretolerance to external antigens can be created by manipulating the immunesystem. It occurs in three forms: central tolerance, peripheraltolerance and acquired tolerance. Without wishing to be bound by asingle theory, tolerance employs regulatory immune cells—includingTregs—that directly suppress autoreactive cells, as well as severalother immune cell subsets with immunoregulatory properties—includingCD8⁺ T cells and other types of CD4⁺ T cells (Tr1, Th3), in addition tonatural killer (NK), NKT cells, dendritic cells (DC) and B cells.

Tolerance can be induced by blocking costimulation or upon engagement ofa co-inhibitory B7 with its counter receptor. Transfer of toleranceinvolves isolation of the cells that have been induced for toleranceeither in vivo (i.e. prior to cell isolation) or ex-vivo, enrichment andexpansion of these cells ex vivo, followed by reinfusion of the expandedcells to the patient. This method can be used for treatment ofautoimmune diseases as recited herein, immune related diseases asrecited herein, transplantation and graft rejection. Thus, according toat least some embodiments, the invention provides methods for toleranceinduction, comprising in vivo or ex vivo treatment administration ofeffective amount of any one of isolated soluble LY6G6F, VSIG10, TMEM25,LSR polypeptide, or a polypeptide comprising the extracellular domain ofLY6G6F, VSIG10, TMEM25, LSR, or fragment thereof, or a fusion thereof toa heterologous sequence, and/or a polyclonal or monoclonal antibody orantigen binding fragments and conjugates containing same, and/oralternative scaffolds, specific to any one of LY6G6F, VSIG10, TMEM25and/or LSR proteins, to a patient or to leukocytes isolated from thepatient, in order to induce differentiation of tolerogenic regulatorycells, followed by ex-vivo enrichment and expansion of said cells andreinfusion of the tolerogenic regulatory cells to said patient.

According to at least some embodiments, the invention provides assaysfor detecting the presence of LY6G6F, VSIG10, TMEM25 and/or LSR proteinsin vitro or in vivo in a biological sample or an individual, comprisingcontacting the sample with an antibody and/or antigen binding fragmentsand/or conjugates containing same, and/or alternative scaffolds, havingspecificity for LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, anddetecting the binding of LY6G6F, VSIG10, TMEM25 and/or LSR protein inthe sample and/or in the individual.

According to at least some embodiments, there is provided an assay fordetecting the presence of any one of the polypeptides of any of SEQ IDNOs:1-8, 11-18, 47-50, 58, 143, or a variant thereof that is at least95% identical thereto, in a sample.

According to at least some embodiments, there is provided a method fordiagnosing a disease in a subject, comprising detecting in the subjector in a sample obtained from said subject any one of the polypeptides ofany of SEQ ID NOs:1-8, 11-18, 47-50, 58, 143, or a variant thereof thatis at least 95% identical thereto, or fragments thereof.

Optionally detecting the polypeptide is performed in vivo or in vitro.

Optionally the detection is conducted by immunoassay.

Optionally the detection is conducted using antibodies or fragments asdescribed herein.

According to at least some embodiments, the invention provides methodsfor detecting a disease, diagnosing a disease, monitoring diseaseprogression or treatment efficacy or relapse of a disease, or selectinga therapy for a disease, detect cells affected by the foregoing disease,comprising detecting expression of a LY6G6F, VSIG10, TMEM25 and/or LSR,wherein the disease is selected from cancer, infectious disorder asrecited herein, and/or immune related disorder.

According to one embodiment, detecting the presence of the polypeptideis indicative of the presence of the disease and/or its severity and/orits progress. According to another embodiment, a change in theexpression and/or the level of the polypeptide compared to itsexpression and/or level in a healthy subject or a sample obtainedtherefrom is indicative of the presence of the disease and/or itsseverity and/or its progress. According to a further embodiment, achange in the expression and/or level of the polypeptide compared to itslevel and/or expression in said subject or in a sample obtainedtherefrom at earlier stage is indicative of the progress of the disease.According to still further embodiment, detecting the presence and/orrelative change in the expression and/or level of the polypeptide isuseful for selecting a treatment and/or monitoring a treatment of thedisease.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C-1 to 1C-3, 1D-1H, 1I-1, 1I-2, 1J-1 and 1J-2 presentamino acid sequences of LY6G6F (FIG. 1A, SEQ ID NO:1), VSIG10 (FIGS. 1B,SEQ ID NO:3, and 1C, SEQ ID NO:5), TMEM25 (FIGS. 1D, SEQ ID NO:7), LSR(FIGS. 1E (SEQ ID NO:11), 1F (SEQ ID NO:13), 1G (SEQ ID NO:15), 1H (SEQID NO:16), 1I (SEQ ID NO:17), and 1J (SEQ ID NO:18)) proteins,fragments, ECDs and the corresponding nucleic acid sequences encodingsame Amino acid residues corresponding to signal peptide (SP) appear inbold Italics. Ig-V and/or Ig-C domains are shown in boxes Amino acidresidues corresponding to thransmembrane region (TM) appear in bold andunderlined Amino acid residues corresponding to alternative exonsskipping in some of the isoforms (in FIGS. 1B, and 1E) appear in Italicsand underlined. Nucleic acid sequence corresponding to alternative exonsskipping variants of VSIG10 (skipping exon 3), and LSR (isoform-e,skipping exons 3, 4 and 5) appears in bold in FIGS. 1C, and 1I,respectively. Nucleic acid sequence corresponding to transmembraneregion (TM) appears in bold and underlined in FIG. 1C. Nucleic acidsequence corresponding to signal peptide (SP) appears in bold Italics inFIGS. 1C, 1E, 1G, 1H, 1I, and 1J. TGA stop codon is highlighted in FIGS.1C, and H.

FIGS. 2A-1, 2A-2, 2B-1(1), 2B-1(2), 2B-2(1), 2B-2(2), 2C-1(1), 2C-1(2),2C-2(1), 2C-2(2), 2D-1(1), 2D-1(2), 2D-2(1), 2D-2(2), 2E-1(1), 2E-1(2),2E-2(1), 2E-2(2), 2F-1, 2F-2, 2G-1(1), 2G-1(2), 2G-2(1) and 2G-2(2)present amino acid sequence comparison between: the VSIG10 variant SEQID NO:5 and the known VSIG10 protein, SEQ ID NO: 3 (genbank accessionnumber NP_061959.2) (FIG. 2A); LSR_isoform-a, SEQ ID NO:11 and known LSRprotein, genbank accession number NP_991403 SEQ ID NO:62 (FIG. 2B-1);LSR_isoform-a, SEQ ID NO:11 and known LSR protein, genbank accessionnumber XP_002829104, SEQ ID NO:68 (FIG. 2B-2); LSR_isoform-b, SEQ IDNO:13 and known LSR protein, genbank accession number NP_057009, SEQ IDNO:63 (FIG. 2C-1); LSR_isoform-b, SEQ ID NO:13 and known LSR protein,genbank accession number BAC11614, SEQ ID NO:65 (FIG. 2C-2);LSR_isoform-c, SEQ ID NO:15 and known LSR protein, genbank accessionnumber NP_991404, SEQ ID NO:66 (FIG. 2D-1); LSR_isoform-c, SEQ ID NO:15and known LSR protein, genbank accession number XP_002829105.1, SEQ IDNO:69 (FIG. 2D-2); LSR_isoform-d, SEQ ID NO:16 and known LSR protein,genbank accession number NP_991404, SEQ ID NO:66 (FIG. 2E-1);LSR_isoform-d, SEQ ID NO:16 and known LSR protein, genbank accessionnumber XP_002829105.1, SEQ ID NO:69 (FIG. 2E-2); LSR_isoform-e, SEQ IDNO:17 and known LSR protein, genbank accession number BAG59226.1, SEQ IDNO:67 (FIG. 2F); LSR_isoform-f, SEQ ID NO:18 and known LSR protein,genbank accession number NP_991403, SEQ ID NO:62 (FIG. 2G-1);LSR_isoform-f, SEQ ID NO:18 and known LSR protein, genbank accessionnumber NP_991404, SEQ ID NO:66 (FIG. 2G-2). The sequence of the uniqueedge portions (unique junction) of the VSIG10 variant (SEQ ID NO:5) andLSR variant (SEQ ID NO:18) are bold and highlighted (FIGS. 2A and 2G,respectively).

FIGS. 3A and 3B show scatter plots, demonstrating the expression ofVSIG10 transcripts, that encode the VSIG10 proteins, on a virtual panelof all tissues and conditions using MED discovery engine, demonstratingdifferential expression of VSIG10 transcripts in several groups of cellsfrom the immune system, mainly in leukocytes, and in various cancerconditions, such as CD10+ leukocytes from ALL and BM-CD34+cells fromAML.

FIGS. 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1, 4D-2 and 4E show scatterplots, demonstrating the expression of LSR transcripts, that encode theLSR proteins, on a virtual panel of all tissues and conditions using MEDdiscovery engine, demonstrating differential expression of LSRtranscripts in several groups of cells from the immune system, mainly inbone marrow cells, and in various cancerous conditions of tissues, suchas in breast, lung, ovary, pancreas, prostate and skin cancers.

FIG. 5A presents LY6G6F human (SEQ ID NO: 1) and mouse(refINP_001156664.1, SEQ ID NO:29) amino acid sequence comparison. FIGS.5B-1, 5B-2 present VSIG10 human (SEQ ID NO: 3) and mouse (spID3YX43.2,SEQ ID NO:30) amino acid sequence comparison. FIGS. 5C-1 to 5C-4 presentLSR human (SEQ ID NO:11) and either mouse (refINP_059101.1, SEQ IDNO:31) or mouse (refINP_001157656.1, SEQ ID NO:32) amino acid sequencecomparison. FIG. 5D presents TMEM25 human (SEQ ID NO:7) and mouse (ref:1c114109, SEQ ID NO:28) amino acid sequence comparison.

FIG. 6 presents a table summarizing the primers which were used forcloning of LY6G6F transcript fused to EGFP. Gene specific sequences areshown in bold face; the restriction site extensions utilized for cloningpurposes are in Italic; and Kozak sequence are underlined.

FIGS. 7-1 and 7-2 present the DNA sequence of LY6G6F full length_fusedto EGFP. The gene specific sequence corresponding to the LY6G6F fulllength sequence is marked in bold faced, EGFP sequence is unbold Italicunderline.

FIG. 8 presents the amino acid sequence of the resulting LY6G6F fulllength fused to EGFP. The gene specific sequence corresponding to thefull length sequence of LY6G6F is marked in bold faced; EGFP sequence isunbold Italic underline.

FIG. 9 presents cell localization of G6F_EGFP fusion protein transientlyexpressed in HEK293T cells. The image was obtained using the 40×objective of the confocal microscope.

FIGS. 10 and 10A-10D present mouse ECDs fused to mouse IgG2a Fc asfollows: mouse LY6G6F (also referred to herein as LY6G6F-Ig, FIG. 10A),mouse VSIG10 (FIG. 10B), mouse TMEM25 (also referred to herein asTMEM25-Ig, FIG. 10C) or mouse LSR (also referred to herein as LSR-Ig,FIG. 10D) ECD-mIgG2aFc fused proteins (SEQ ID NOs: 23, 24, 25, or 26,respectively) Amino acid residues corresponding to signal peptide (SP)are shown in Italics. Amino acid residues corresponding to ECD sequenceare underlined Amino acid residues corresponding to mouse IgG2a Fc areshown in bold face (SEQ ID NO:27).

FIGS. 11A-11J present amino acid sequences of human ECDs fused to humanIgG1 Fc with the Cys at position 220 (according to full length humanIgG1, position 5 in SEQ ID NO:70) replaced with a Ser (SEQ ID NO:156),as follows: human LY6G6F (FIG. 11A), human VSIG10 (FIG. 11B), humanVSIG10-skipping exon 3 variant (FIG. 11C), human TMEM25 (FIG. 11D),human LSR isoform a (FIG. 11E), human LSR isoform b (FIG. 11F), humanLSR isoform c (FIG. 11G), human LSR isoform d (FIG. 11H), human LSRisoform e (FIG. 11I), human LSR isoform f (FIG. 11J) ECD fused to humanIgG1 Fc (SEQ ID NOs: 71-80, respectively) Amino acid residuescorresponding to signal peptide (SP) are shown in bold Italics. Aminoacid residues corresponding to human ECD sequence are underlined Aminoacid residues corresponding to human IgG1 Fc with the Cys at position220 replaced with a Ser (SEQ ID NO:156) are unmarked.

FIGS. 12A-12C are histograms showing over expression of the LSRtranscripts detectable by or according toLSR_seg24-36_200-309/310_Amplicon (SEQ ID:140) in cancerous ovarysamples relative to the normal samples.

FIGS. 13A-13C are histograms showing over expression of the LSRtranscripts detectable by or according toLSR_seg24-36_200-309/310_Amplicon (SEQ ID:140) in cancerous breastsamples relative to the normal samples.

FIGS. 14A, 14B are histograms showing over expression of the LSRtranscripts detectable by or according toLSR_seg24-36_200-309/310_Amplicon (SEQ ID:140) in cancerous lung samplesrelative to the normal samples.

FIGS. 15A, 15B are histograms showing over expression of the LSRtranscripts detectable by or according toLSR_seg24-36_200-309/310_Amplicon (SEQ ID:140) in normal tissue samplesrelative to the ovary samples.

FIG. 16 is a histogram showing over expression of the LSR transcriptsdetectable by or according to LSR_seg24-36_200-309/310_Amplicon (SEQID:140) in cancerous kidney samples relative to the normal samples.

FIG. 17 is a histogram showing over expression of the LSR transcriptsdetectable by or according to LSR_seg24-36_200-309/310_Amplicon (SEQID:140) in cancerous liver samples relative to the normal samples.

FIG. 18 demonstrates Western Blot analysis of the expression ofLSR_P5a_Flag_m protein (SEQ ID: 144) in stably-transfected recombinantHEK293T cells, as detected with anti Flag (Sigma cat#A8592) (FIG. 18A)and anti LSR antibodies as follow: Abnova, cat#H00051599-B01P (FIG. 18B)Abcam, cat ab59646 (FIG. 18C) and Sigma cat# HPA007270 (FIG. 18D). Lane1: HEK293T_pIRESpuro3; lane 2: HEK293T_pIRESpuro3_LSR_P5a_Flag.

FIGS. 19A-19D demonstrate the subcellular localization ofLSR_P5a_Flag_m. LSR_P5a_Flag_m (SEQ ID NO: 144) is localized mainly tothe cell cytoplasm, but can also be detected on the cell surface asdetected with anti Flag (Sigma cat# A9594) (FIG. 19A) and anti LSRantibodies as follows: Abcam, cat ab59646 (FIG. 19B) Abnova,cat#H00051599-B01P (FIG. 19C) and Sigma cat# HPA007270 (FIG. 19D).

FIG. 20 demonstrates the endogenous expression of LSR in various celllines. A band at 72 kDa corresponding to LSR was detected with anti LSRantibody in extracts of (1) Caov3, (2) ES2, (3) OV-90, (4) OVCAR3, (5)SK-OV3, (6) TOV112D, (7) CaCo2, (8) HeLa, (9) Hep G2, (10) MCF-7, (11)SkBR3 and (12) 293T_LSR_P5a_Flag (FIG. 20A). Anti GAPDH (Abcam cat#ab9484) served as a loading control (FIG. 20B).

FIGS. 21A-21C are histogram showing expression of TMEM25 transcriptsdetectable by or according toseg21-27—TMEM25_seg_21-27_200-344/346_Amplicon (SEQ ID NO: 123) innormal and cancerous Breast tissues.

FIGS. 22A-22C are histograms showing expression of TMEM25 transcriptsdetectable by or according toseg21-27—TMEM25_seg_21-27_200-344/346_Amplicon (SEQ ID NO: 123) indifferent normal tissues.

FIGS. 23A, 23B demonstrate Western blot results showing (A) specificinteraction between Rabbit anti TMEM25 antibodies and TMEM25_P5 protein(SEQ ID NO: 7) and TMEM25_P5_Flag (SEQ ID NO: 129), but not HEK_293T_pRp3. (B) specific interaction between TMEM25_P5_Flag protein (SEQ IDNO: 129) and anti-Flag antibodies. Lane1: HEK293T_pIRESpuro3; lane 2:HEK293T_pIRESpuro3_TMEM25-P5, lane 3: HEK293T_pIRESpuro3_TMEM25-P5-Flag.

FIGS. 24A-24C present the cell surface localization of TMEM25_P5 (SEQ IDNO:132) (FIG. 24A) and TMEM25_P5_Flag (SEQ ID NO: 129) (FIG. 24B) usinganti TMEM25 Abs. FIG. 24C demonstrate TMEM25_P5_Flag (SEQ ID NO: 129)localization using anti flag Abs (Sigma, catalog number: A9594).

FIG. 25 demonstrates that anti TMEM25 antibodies bind to the full lengthTMEM25 protein, in HEK293T recombinant cells expressing TMEM25_P5_Flagprotein (1:2250) (FIG. 25A), as compared to mouse serum (1:2250) (FIG.25B) used as a negative control, indicating membrane localization ofTMEM25 protein.

FIG. 26 presents Western Blot results showing the expression ofendogenous TMEM25 protein in various cell lines: (1):HEK293T_pIRESpuro3, (2) HEK293T_pIRESpuro3_TMEM25-P5-Flag, (3) KARPAS,(4) G-361, (5) RPMI8226, (6) DAUDI, (7) Jurkat.

FIG. 27 demonstrates specific knockdown of TMEM25_P5_Flag protein (SEQID NO: 129) in HEK293T cells stably expressing TMEM25_P5_Flag (SEQ ID NO129) transfected with TMEM25_P5 siRNA (L-018183-00-0005, Dharmacon)(Lane 2) compared to HEK293T cells stably expressing TMEM25_P5_FLAGtransfected with Scrambled-SiRNA (Lane 1) (Dharmacon, D-001810-10-05),using anti TMEM25 antibodies (Sigma, cat# HPA012163).

FIGS. 28A-28F demonstrate that anti LSR (Cat no. ab59646, Abcam) insections of positive control cell line (LSR_P5a_Flag_m transfectedHEK293T cells (coloumn 1, panels A, C and E) shows specificimmunoreactivity in a dose dependent concentrations of 3, 1 and 0.3ug/ml respectively, as compared to the negative control cell line emptyvector HEK293T cells (coloumn 2, panels B, D and F), in pH 9 antigenretrieval method.

FIGS. 29A-29F demonstrate that anti TMEM25 (Cat no. HPA012163, Sigma) insections of positive control cell line TMEM25_P5_Flag transfectedHEK293T cells (coloumn 1, panels A, C and E) shows specificimmunoreactivity in a dose dependent concentrations of 3, 1 and 0.3ug/ml respectively, as compared to the negative control cell line emptyvector HEK293T cells (coloumn 2 panels B, D and F), in pH 9 antigenretrieval method.

FIGS. 30A-30E show the in vitro inhibitory effect of soluble LY6G6F-Ig(SEQ ID NO:23), TMEM25-Ig (SEQ ID NO:25) and LSR-Ig (SEQ ID NO:26) onmouse T cells activation. Activation of T cells isolated from spleens ofD011.10 mice was induced with 20 ug/ml (FIGS. 30A-C, E) or 2 ug/ml(Figures D and F) OVA323-339 in the presence of irradiated splenocytedfrom Balb/c mice that serve as APCs. In these studies CTLA4-Ig orB7-H4-Ig were used as positive controls while mouse IgG2a was used as Igcontrol.

FIG. 31 shows the in vitro inhibitory effect of bead bound LSR-Ig (SEQID NO:26) on T cell proliferation induced by anti-CD3 and anti-CD28coated beads.

FIGS. 32A and 32B show the effect of LY6G6F, VSIG10, TMEM25 and LSRfusion proteins (SEQ ID NO:23-26, respectively) on CD4 T cellactivation, as manifested by reduced IFNγ secretion (A) and reducedexpression of the activation marker CD69 (B). Each bar is the mean ofduplicate cultures, the error bars indicating the standard deviation(Student t-test,*P<0.05, **p<0.01, compared with control mIgG2a.

FIGS. 33A-33D show the effect of stimulator cells (a murine thymoma cellline, Bw5147, which were engineered to express membrane-bound anti-humanCD3 antibody fragments) expressing the cDNAs encoding human LY6G6F,TMEM25 or LSR (SEQ ID NOs: 1, 7 or 11, respectively) on theproliferation (CPM) of bulk human T cells (FIG. 33A), CD4+ human T cells(FIG. 33B), CD8+ human T cells (FIG. 33C), or naïve CD4CD45RA+ human Tcells (FIG. 33D). Results are displayed as the mean+/−SEM of 6 (FIG.337A) or 3 (FIGS. 33B, C, and D) experiments. *P<0.05, **p<0.01,***p<0.001, and #p<0.0001 (Students T-test) represent significantlydifferent results compared to empty vector.

FIGS. 34A, 34B show the therapeutic effect of LSR-Ig (SEQ ID NO:26) orTMEM25-Ig (SEQ ID NO:25) treatment in the PLP139-151-induced R-EAE modelin SJL mice. LSR-Ig (SEQ ID NO:26) or TMEM25-Ig (SEQ ID NO:25) wereadministered in a therapeutic mode from the onset of disease remission(day 18), at 100 microg/mouse i.p. 3 times per week for two weeks.Therapeutic effects of LSR-Ig and TMEM25-Ig on clinical symptoms aredemonstrated as reduction in Mean Clinical Score (FIG. 34A). Inaddition, LSR-Ig and TMEM25-Ig treatment inhibited DTH responses toinducing epitope (PLP139-151) or spread epitope (PLP178-191), on day 35after R-EAE induction (FIG. 34B). In this study the effect of LSR-Ig orTMEM25-Ig was studied in comparison to mIgG2a Ig negative control andCTLA4-Ig positive control that were administered at a similar regimen asthe test proteins.

FIGS. 35A-35E show the dose dependency and mode of action of the effectof TMEM25-Ig (SEQ ID NO:25) in the R-EAE model in SJL mice. In thisstudy, treatments were given from onset of disease remission (day 19) at100, 30 or 10 microg/mouse i.p. 3 times per week for two weeks, ascompared to 100 microg/mouse IgG2a control that was given at a similarschedule. shown are effects of TMEM25-Ig treatment on disease course(FIG. 35A), DTH responses to spread epitopes PLP178-191 and MBP84-104 ondays 45 and 76 post R-EAE induction (FIG. 35B), ex-vivo recall responsesof splenocytes isolated on day 45 and 75 post disease induction (FIG.35C) and LN cells isolated on day 45 post disease induction (FIG. 35D)as manifested by the effect of TMEM25-Ig treatment on cell proliferationand cytokine secretion (IFNg, IL-17, IL-10 and IL-4). The effect ofTMEM25-Ig on cell counts in the spleen, lymph nodes and CNS as well asthe different linages present in the CNS upon treatment with TMEM25-Igat 100 ug/dose is shown in FIG. 35E.

FIGS. 36A-36E show the therapeutic effect of VSIG10-Ig (SEQ ID NO:24)treatment in the PLP139-151-induced R-EAE model in SJL mice. VSIG10-Ig(SEQ ID NO:24) was administered in a therapeutic mode from the onset ofdisease remission (day 19), at 100 microg/mouse i.p. 3 times per weekfor two weeks. Therapeutic effects of VSIG10-Ig on clinical symptoms isdemonstrated as reduction in Mean Clinical Score (FIG. 36A). Inaddition, VSIG10-Ig treatment inhibited DTH responses to spread epitopes(PLP178-191 and MBP MBP84-104), on days 45 and 76 after R-EAE induction(FIG. 36B). Also shown is the effect of VSIG10-Ig on ex-vivo recallresponses of splenocytes isolated on day 45 and 75 post diseaseinduction (FIG. 36C) and LN cells isolated on day 45 post diseaseinduction (FIG. 36D) as manifested by the effect of VSIG10-Ig treatmenton cell proliferation and cytokine secretion (IFNg, IL-17, IL-10 andIL-4). The effect of VSIG10-Ig on cell counts in the spleen, lymph nodesand CNS as well as the different linages present within each of thesetissues upon treatment with VSIG10-Ig at 100 ug/dose is shown in FIG.36E. In this study the effect of VSIG10-Ig was studied in comparison tomIgG2a Ig control that was administered at similar dose and regimen asVSIG10-Ig.

FIGS. 37A-37C show the therapeutic effect of LSR-Ig (SEQ ID NO:26)administered at 100 microg/mouse, i.p, 3 times per week for 10 days incollagen induced arthritis (CIA) model of Rheumatoid Arthritis. Measuredare clinical score (A) paw swelling (B) and histological damage (C)CTLA4-Ig, (100 microg/mouse) and TNFR-Ig (etanercept) were used as apositive control while mIgG2a Ig control (100 microg/mouse) was used asnegative control.

FIG. 38 shows the therapeutic effect of LY6G6F-Ig (SEQ ID NO:23)administered at 25 mg/kg, i.p, 3 times per week for 2 weeks in collageninduced arthritis (CIA) model of Rheumatoid Arthritis, with measurementsgiven according to clinical scores.

For FIGS. 12-17, 21, 22, division was made into separate parts “A”, “B”and so forth for reasons of space only, so as to be able to show allresults.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in at least some embodiments, relates to any oneof the proteins referred to as LY6G6F, VSIG10, TMEM25 and/or LSR, andits corresponding nucleic acid sequence, and portions and variantsthereof and fusion proteins and conjugates containing, and/or polyclonaland monoclonal antibodies and/or antigen binding fragments and/orconjugates containing same, and/or alternative scaffolds thereof thatbind LY6G6F, VSIG10, TMEM25 and/or LSR and/or portions and/or variantsthereof, and the use thereof as a therapeutic and/or diagnostic agent,and various uses as described herein.

US Patent Application Nos. US2009117566, US20090017473, and other familymembers, assigned to GENENTECH INC., disclose a 382 amino acid LY6G6Fprotein sequence (DNA234441, tumor-associated antigenic target (TAT)TAT201, SEQ ID NO:92 therein) having a transmembrane domain betweenresidues 234-254 and 354-374. '566, '473, applications and otherapplications from this patent family disclose that TAT201 is overexpressed in colon and rectal cancers. PCT Application Nos WO2003083074and WO2004046342 disclose a 382 amino acid LY6G6F protein sequence asone of many genes that are over expressed in colon cancer cells. Thesepatent applications further purportedly relate to methods of use ofLY6G6F for detecting and treating colon cancer. However, these patentapplications do not teach or suggest or provide any incentive that woulddirect a skilled artisan to use antibodies specific to the LY6G6F and/orLY6G6F ECD for treatment and/or diagnosis of cancer other thancolorectal cancer, and/or infectious disorders, and/or immune relateddisorders. These patent applications do not describe LY6G6F ECD and donot teach or suggest or provide any incentive that would direct askilled artisan to use the LY6G6F ECD for treatment of cancer and/orinfectious disorders, and/or immune related disorders.

TMEM25 is disclosed in PCT Application Nos WO9958642 and WO2003087300,and US Patent Application Nos. US2007041963 and US2005202526, as one ofmany (hundreds to thousands) proteins, useful for diagnosing,preventing, and treating disorders associated with an abnormalexpression or activity of these proteins. However, these applications donot teach or suggest or provide any incentive that would direct askilled artisan to use antibodies specific to the TMEM25 and/or TMEM25ECD for treatment and/or diagnosis of cancer and/or infectiousdisorders, and/or immune related disorders. TMEM25 is also disclosed inUS Patent Application No. US2004010134, as one of hundreds of albuminfusion proteins, useful for diagnosing, treating, preventing orameliorating diseases or disorders e.g. cancer, anemia, arthritis,asthma, inflammatory bowel disease or Alzheimer's disease. However, thisapplication does not teach or suggest or provide any incentive thatwould direct a skilled artisan to use antibodies specific to the TMEM25and/or TMEM25 ECD for treatment and/or diagnosis of cancer and/orinfectious disorders, and/or immune related disorders. TMEM25 is alsodescribed in Doolan P, et al., Tumour Biol. 2009, 30(4):200-9 as afavourable prognostic and predictive biomarker for breast cancerdiagnosis. However, this publication does not teach or suggest orprovide any incentive that would direct a skilled artisan to use theantibodies specific to TMEM25 and/or TMEM25 ECD for treatment of cancerand/or infectious disorders, and/or immune related disorders.

In order that the present invention in various embodiments may be morereadily understood, certain terms are first defined. Additionaldefinitions are set forth throughout the detailed description.

As used herein the term “isolated” refers to a compound of interest (forexample a polynucleotide or a polypeptide) that is in an environmentdifferent from that in which the compound naturally occurs e.g.separated from its natural milieu such as by concentrating a peptide toa concentration at which it is not found in nature. “Isolated” includescompounds that are within samples that are substantially enriched forthe compound of interest and/or in which the compound of interest ispartially or substantially purified.

An “immune cell” refers to any cell from the hemopoietic originincluding but not limited to T cells, B cells, monocytes, dendriticcells, and macrophages.

As used herein, the term “polypeptide” refers to a chain of amino acidsof any length, regardless of modification (e.g., phosphorylation orglycosylation).

As used herein, a “costimulatory polypeptide” or “costimulatorymolecule” is a polypeptide that, upon interaction with a cell-surfacemolecule on T cells, modulates T cell responses.

As used herein, a “costimulatory signaling” is the signaling activityresulting from the interaction between costimulatory polypeptides onantigen presenting cells and their receptors on T cells duringantigen-specific T cell responses. Without wishing to be limited by asingle hypothesis, the antigen-specific T cell response is believed tobe mediated by two signals: 1) engagement of the T cell Receptor (TCR)with antigenic peptide presented in the context of MHC (signal 1), and2) a second antigen-independent signal delivered by contact betweendifferent costimulatory receptor/ligand pairs (signal 2). Withoutwishing to be limited by a single hypothesis, this “second signal” iscritical in determining the type of T cell response (activation vsinhibition) as well as the strength and duration of that response, andis regulated by both positive and negative signals from costimulatorymolecules, such as the B7 family of proteins.

As used herein, the term “B7” polypeptide means a member of the B7family of proteins that costimulate T cells including, but not limitedto B7-1, B7-2, B7-DC, B7-H5, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-S3and biologically active fragments and/or variants thereof.Representative biologically active fragments include the extracellulardomain or fragments of the extracellular domain that costimulate Tcells.

As used herein, a “variant” polypeptide contains at least one amino acidsequence alteration as compared to the amino acid sequence of thecorresponding wild-type polypeptide.

As used herein, “conservative” amino acid substitutions aresubstitutions wherein the substituted amino acid has similar structuralor chemical properties. As used herein, the term “host cell” refers toprokaryotic and eukaryotic cells into which a recombinant vector can beintroduced.

As used herein, the term “an edge portion” or “a new junction” refers toa connection between two portions of a splice variant according to thepresent invention that were not joined in the wild type or knownprotein. An edge may optionally arise due to a join between the above“known protein” portion of a variant and the tail, for example, and/ormay occur if an internal portion of the wild type sequence is no longerpresent, such that two portions of the sequence are now contiguous inthe splice variant that were not contiguous in the known protein. A“bridge” may optionally be an edge portion as described above, but mayalso include a join between a head and a “known protein” portion of avariant, or a join between a tail and a “known protein” portion of avariant, or a join between an insertion and a “known protein” portion ofa variant.

In some embodiments, a bridge between a tail or a head or a uniqueinsertion, and a “known protein” portion of a variant, comprises atleast about 10 amino acids, or in some embodiments at least about 20amino acids, or in some embodiments at least about 30 amino acids, or insome embodiments at least about 40 amino acids, in which at least oneamino acid is from the tail/head/insertion and at least one amino acidis from the “known protein” portion of a variant. In some embodiments,the bridge may comprise any number of amino acids from about 10 to about40 amino acids (for example, 10, 11, 12, 13 . . . 37, 38, 39, 40 aminoacids in length, or any number in between).

It should be noted that a bridge cannot be extended beyond the length ofthe sequence in either direction, and it should be assumed that everybridge description is to be read in such manner that the bridge lengthdoes not extend beyond the sequence itself.

Furthermore, bridges are described with regard to a sliding window incertain contexts below. For example, certain descriptions of the bridgesfeature the following format: a bridge between two edges (in which aportion of the known protein is not present in the variant) mayoptionally be described as follows: a bridge portion of CONTIG-NAME_P1(representing the name of the protein), comprising a polypeptide havinga length “n”, wherein n is at least about 10 amino acids in length,optionally at least about 20 amino acids, at least about 30 amino acids,at least about 40 amino acids, or at least about 50 amino acids inlength, wherein at least two amino acids comprise XX (2 amino acids inthe center of the bridge, one from each end of the edge), having astructure as follows (numbering according to the sequence ofCONTIG-NAME_P1): a sequence starting from any of amino acid numbers 49-xto 49 (for example); and ending at any of amino acid numbers50+((n−2)−x) (for example), in which x varies from 0 to n−2. In thisexample, it should also be read as including bridges in which n is anynumber of amino acids between 10-50 amino acids in length. Furthermore,the bridge polypeptide cannot extend beyond the sequence, so it shouldbe read such that 49-x (for example) is not less than 1, nor50+((n−2)−x) (for example) greater than the total sequence length.

The term “cancer” as used herein should be understood to encompass anyneoplastic disease (whether invasive or metastatic) which ischaracterized by abnormal and uncontrolled cell division causingmalignant growth or tumor. Non-limiting examples of cancer which may betreated with a compound according to at least some embodiments of thepresent invention are solid tumors, sarcomas and hematologicalmalignancies, including but not limited to breast cancer (e.g. breastcarcinoma), cervical cancer, ovary cancer (ovary carcinoma), endometrialcancer, melanoma, bladder cancer (bladder carcinoma), lung cancer (e.g.adenocarcinoma and non-small cell lung cancer), pancreatic cancer (e.g.pancreatic carcinoma such as exocrine pancreatic carcinoma), coloncancer (e.g. colorectal carcinoma, such ascolon adenocarcinoma and colonadenoma), prostate cancer including the advanced disease, hematopoietictumors of lymphoid lineage (e.g. leukemia, acute lymphocytic leukemia,chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma,multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma), myeloidleukemia (for example, acute myelogenous leukemia (AML), chronicmyelogenous leukemia), thyroid cancer, thyroid follicular cancer,myelodysplastic syndrome (MDS), tumors of mesenchymal origin (e.g.fibrosarcomas and rhabdomyosarcomas), melanoma, uveal melanoma,teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor ofthe skin (e.g. keratoacanthomas), renal cancer, anaplastic large-celllymphoma, esophageal squamous cells carcinoma, hepatocellular carcinoma,follicular dendritic cell carcinoma, intestinal cancer, muscle-invasivecancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer,bladder cancer, head and neck cancer, stomach cancer, liver cancer, bonecancer, brain cancer, cancer of the retina, biliary cancer, small bowelcancer, salivary gland cancer, cancer of uterus, cancer of testicles,cancer of connective tissue, prostatic hypertrophy, myelodysplasia,Waldenstrom's macroglobinaemia, nasopharyngeal, neuroendocrine cancer,myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma,carcinoid, oesophagogastric, fallopian tube cancer, peritoneal cancer,papillary serous mullerian cancer, malignant ascites, gastrointestinalstromal tumor (GIST), and a hereditary cancer syndrome such asLi-Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL), and whereinthe cancer may be non-metastatic, invasive or metastatic.

According to at least some preferred embodiments of the presentinvention, the cancer is selected from the group consisting of melanoma,cancers of liver, renal, brain, breast, colon, lung, ovary, pancreas,prostate, stomach, multiple myeloma and hematopoietic cancer, includingbut not limited to lymphoma (Hodgkin's and non Hodgkin's), acute andchronic lymphoblastic leukemia and acute and chronic myeloid leukemia,and wherein the cancer may be non-metastatic, invasive or metastatic.

The term “autoimmune disease” as used herein should be understood toencompass any autoimmune disease and chronic inflammatory conditions.According to at least some embodiments of the invention, the autoimmunediseases should be understood to encompass any disease disorder orcondition selected from the group including but not limited to multiplesclerosis, including relapsing-remitting multiple sclerosis, primaryprogressive multiple sclerosis, and secondary progressive multiplesclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis,systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease;benign lymphocytic angiitis, thrombocytopenic purpura, idiopathicthrombocytopenia, idiopathic autoimmune hemolytic anemia, pure red cellaplasia, Sjogren's syndrome, rheumatic disease, connective tissuedisease, inflammatory rheumatism, degenerative rheumatism,extra-articular rheumatism, juvenile rheumatoid arthritis, arthritisuratica, muscular rheumatism, chronic polyarthritis, cryoglobulinemicvasculitis, ANCA-associated vasculitis, antiphospholipid syndrome,myasthenia gravis, autoimmune haemolytic anaemia, Guillian-Barresyndrome, chronic immune polyneuropathy, autoimmune thyroiditis, insulindependent diabetes mellitus, type I diabetes, Addison's disease,membranous glomerulonephropathy, Goodpasture's disease, autoimmunegastritis, autoimmune atrophic gastritis, pernicious anaemia, pemphigus,pemphigus vulgarus, cirrhosis, primary biliary cirrhosis,dermatomyositis, polymyositis, fibromyositis, myogelosis, celiacdisease, immunoglobulin A nephropathy, Henoch-Schonlein purpura, Evanssyndrome, atopic dermatitis, psoriasis, psoriasis arthropathica, Graves'disease, Graves' ophthalmopathy, scleroderma, systemic scleroderma,progressive systemic scleroderma, asthma, allergy, primary biliarycirrhosis, Hashimoto's thyroiditis, primary myxedema, sympatheticophthalmia, autoimmune uveitis, hepatitis, chronic action hepatitis,collagen diseases, ankylosing spondylitis, periarthritishumeroscapularis, panarteritis nodosa, chondrocalcinosis, Wegener'sgranulomatosis, microscopic polyangiitis, chronic urticaria, bullousskin disorders, pemphigoid, atopic eczema, Devic's disease, childhoodautoimmune hemolytic anemia, Refractory or chronic AutoimmuneCytopenias, Prevention of development of Autoimmune Anti-Factor VIIIAntibodies in Acquired Hemophilia A, Cold Agglutinin Disease,Neuromyelitis Optica, Stiff Person Syndrome, gingivitis, periodontitis,pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis,and inflammatory skin disorders, selected from the group consisting ofpsoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne,normocomplementemic urticarial vasculitis, pericarditis, myositis,anti-synthetase syndrome, scleritis, macrophage activation syndrome,Bechet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult andjuvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome,familial cold-induced auto-inflammatory syndrome, neonatal onsetmultisystemic inflammatory disease, familial Mediterranean fever,chronic infantile neurologic, cutaneous and articular syndrome, systemicjuvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler'ssyndrome, autoimmune retinopathy, age-related macular degeneration,atherosclerosis, chronic prostatitis and TNF receptor-associatedperiodic syndrome (TRAPS).

Optionally and preferably, the autoimmune disease includes but is notlimited to any of the types and subtypes of any of multiple sclerosis,rheumatoid arthritis, type I diabetes, psoriasis, systemic lupuserythematosus, inflammatory bowel disease, uveitis, or Sjogren'ssyndrome.

As used herein, “multiple sclerosis” comprises one or more of multiplesclerosis, benign multiple sclerosis, relapsing remitting multiplesclerosis, secondary progressive multiple sclerosis, primary progressivemultiple sclerosis, progressive relapsing multiple sclerosis, chronicprogressive multiple sclerosis, transitional/progressive multiplesclerosis, rapidly worsening multiple sclerosis, clinically-definitemultiple sclerosis, malignant multiple sclerosis, also known asMarburg's Variant, and acute multiple sclerosis. Optionally, “conditionsrelating to multiple sclerosis” include, e.g., Devic's disease, alsoknown as Neuromyelitis Optica; acute disseminated encephalomyelitis,acute demyelinating optic neuritis, demyelinative transverse myelitis,Miller-Fisher syndrome, encephalomyelradiculoneuropathy, acutedemyelinative polyneuropathy, tumefactive multiple sclerosis and Balo'sconcentric sclerosis.

As used herein, “rheumatoid arthritis” comprises one or more ofrheumatoid arthritis, gout and pseudo-gout, juvenile idiopathicarthritis, juvenile rheumatoid arthritis, Still's disease, ankylosingspondylitis, rheumatoid vasculitis. Optionally, conditions relating torheumatoid arthritis include, e.g., osteoarthritis, sarcoidosis,Henoch-Schönlein purpura, Psoriatic arthritis, Reactive arthritis,Spondyloarthropathy, septic arthritis, Haemochromatosis, Hepatitis,vasculitis, Wegener's granulomatosis, Lyme disease, FamilialMediterranean fever, Hyperimmunoglobulinemia D with recurrent fever, TNFreceptor associated periodic syndrome, and Enteropathic arthritisassociated with inflammatory bowel disease.

As used herein, “Uveitis” comprises one or more of uveitis, anterioruveitis (or iridocyclitis), intermediate uveitis (pars planitis),posterior uveitis (or chorioretinitis) and the panuveitic form.

As used herein, “inflammatory bowel disease” comprises one or more ofinflammatory bowel disease Crohn's disease, ulcerative colitis (UC),Collagenous colitis, Lymphocytic colitis, Ischaemic colitis, Diversioncolitis, Behçet's disease, Indeterminate colitis.

As used herein, “psoriasis” comprises one or more of psoriasis,Nonpustular Psoriasis including Psoriasis vulgaris and Psoriaticerythroderma (erythrodermic psoriasis), Pustular psoriasis includingGeneralized pustular psoriasis (pustular psoriasis of von Zumbusch),Pustulosis palmaris et plantaris (persistent palmoplantar pustulosis,pustular psoriasis of the Barber type, pustular psoriasis of theextremities), Annular pustular psoriasis, Acrodermatitis continua,Impetigo herpetiformis. Optionally, conditions relating to psoriasisinclude, e.g., drug-induced psoriasis, Inverse psoriasis, Napkinpsoriasis, Seborrheic-like psoriasis, Guttate psoriasis, Nail psoriasis,Psoriatic arthritis.

As used herein, “type 1 diabetes” comprises one or more of type 1diabetes, insulin-dependent diabetes mellitus, idiopathic diabetes,juvenile type ldiabetes, maturity onset diabetes of the young, latentautoimmune diabetes in adults, gestational diabetes. Conditions relatingto type 1 diabetes include, neuropathy including polyneuropathy,mononeuropathy, peripheral neuropathy and autonomicneuropathy; eyecomplications: glaucoma, cataracts, retinopathy.

As used herein, “Sjogren's syndrome” comprises one or more of Sjogren'ssyndrome, Primary Sjogren's syndrome and Secondary Sjogren's syndrome,as well as conditions relating to Sjogren's syndrome includingconnective tissue disease, such as rheumatoid arthritis, systemic lupuserythematosus, or scleroderma. Other complications include pneumonia,pulmonary fibrosis, interstitial nephritis, inflammation of the tissuearound the kidney's filters, glomerulonephritis, renal tubular acidosis,carpal tunnel syndrome, peripheral neuropathy, cranial neuropathy,primary biliary cirrhosis (PBC), cirrhosis, Inflammation in theesophagus, stomach, pancreas, and liver (including hepatitis),Polymyositis, Raynaud's phenomenon, Vasculitis, Autoimmune thyroidproblems, lymphoma.

As used herein, “systemic lupus erythematosus”, comprises one or more ofsystemic lupus erythematosus, discoid lupus, lupus arthritis, lupuspneumonitis, lupus nephritis. Conditions relating to systemic lupuserythematosus include osteoarticular tuberculosis, antiphospholipidantibody syndrome, inflammation of various parts of the heart, such aspericarditis, myocarditis, and endocarditis, Lung and pleurainflammation, pleuritis, pleural effusion, chronic diffuse interstitiallung disease, pulmonary hypertension, pulmonary emboli, pulmonaryhemorrhage, and shrinking lung syndrome, lupus headache,Guillain-Barr{tilde over (e)} syndrome, aseptic meningitis,demyelinating syndrome, mononeuropathy, mononeuritis multiplex,myasthenia gravis, myelopathy, cranial neuropathy, polyneuropathy,vasculitis.

The term “immune related disease (or disorder or condition)” as usedherein should be understood to encompass any disease disorder orcondition selected from the group including but not limited toautoimmune diseases, inflammatory disorders and immune disordersassociated with graft transplantation rejection, such as acute andchronic rejection of organ transplantation, allogenic stem celltransplantation, autologous stem cell transplantation, bone marrowtranplantation, and graft versus host disease.

As used herein the term “inflammatory disorders” and/or “inflammation”,used interchangeably, includes inflammatory abnormalities characterizedby disregulated immune response to harmful stimuli, such as pathogens,damaged cells, or irritants. Inflammatory disorders underlie a vastvariety of human diseases. Non-immune diseases with etiological originsin inflammatory processes include cancer, atherosclerosis, and ischaemicheart disease. Examples of disorders associated with inflammationinclude: Chronic prostatitis, Glomerulonephritis, Hypersensitivities,Pelvic inflammatory disease, Reperfusion injury, Sarcoidosis,Vasculitis, Interstitial cystitis, normocomplementemic urticarialvasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis,macrophage activation syndrome, Bechet's Syndrome, PAPA Syndrome, Blau'sSyndrome, gout, adult and juvenile Still's disease, cryropyrinopathy,Muckle-Wells syndrome, familial cold-induced auto-inflammatory syndrome,neonatal onset multisystemic inflammatory disease, familialMediterranean fever, chronic infantile neurologic, cutaneous andarticular syndrome, systemic juvenile idiopathic arthritis, Hyper IgDsyndrome, Schnitzler's syndrome, TNF receptor-associated periodicsyndrome (TRAPSP), gingivitis, periodontitis, hepatitis, cirrhosis,pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis,and inflammatory skin disorders, selected from the group consisting ofpsoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.

As used herein the term “infectious disorder and/or disease” and/or“infection”, used interchangeably, includes any disorder, disease and/orcondition caused by presence and/or growth of pathogenic biologicalagent in an individual host organism. As used herein the term“infection” comprises the disorder, disease and/or condition as above,exhibiting clinically evident illness (i.e., characteristic medicalsigns and/or symptoms of disease) and/or which is asymtomatic for muchor all of it course. As used herein the term “infection” also comprisesdisorder, disease and/or condition caused by persistence of foreignantigen that lead to exhaustion T cell phenotype characterized byimpaired functionality which is manifested as reduced proliferation andcytokine production. As used herein the term “infectious disorder and/ordisease” and/or “infection”, further includes any of the below listedinfectious disorders, diseases and/or conditions, caused by a bacterialinfection, viral infection, fungal infection and/or parasite infection.

As used herein the term “viral infection” comprises any infection causedby a virus, optionally including but not limited to Retroviridae (e.g.,human immunodeficiency viruses, such as HIV-1 or HIV-2, acquired immunedeficiency (AIDS) also referred to as HTLV-III, LAV or HTLV-III/LAV, orHIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polioviruses, hepatitis A virus; enteroviruses, human coxsackie viruses,rhinoviruses, echoviruses); Calciviridae (e.g., strains that causegastroenteritis); Togaviridae (e.g., equine encephalitis viruses,rubella viruses); Flaviridae (e.g., dengue viruses, encephalitisviruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses);Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses);Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever virus); Reoviridae (e.g., reoviruses, orbiviruses androtaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herperviridae (herpessimplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus(CMV), herpes viruses); Poxviridae (variola virsues, vaccinia viruses,pox viruses); and Iridoviridae (e.g., African swine fever virus); andunclassified viruses (e.g., the etiological agents of Spongiformencephalopathies, the agent of delta hepatitides (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1—internally transmitted; class 2—parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses) as well as Severe acute respiratory syndrome virus andrespiratory syncytial virus (RSV).

As used herein the term “fungal infection” comprises any infectioncaused by a fungi, optionally including but not limited to Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, Candida albicans.

As used herein the term “parasite infection” comprises any infectioncaused by a parasite, optionally including but not limited to protozoa,such as Amebae, Flagellates, Plasmodium falciparum, Toxoplasma gondii,Ciliates, Coccidia, Microsporidia, Sporozoa; helminthes, Nematodes(Roundworms), Cestodes (Tapeworms), Trematodes (Flukes), Arthropods, andaberrant proteins known as prions.

An infectious disorder and/or disease caused by bacteria may optionallycomprise one or more of Sepsis, septic shock, sinusitis, skininfections, pneumonia, bronchitis, meningitis, Bacterial vaginosis,Urinary tract infection (UCI), Bacterial gastroenteritis, Impetigo anderysipelas, Erysipelas, Cellulitis, anthrax, whooping cough, lymedisease, Brucellosis, enteritis, acute enteritis, Tetanus, diphtheria,Pseudomembranous colitis, Gas gangrene, Acute food poisoning, Anaerobiccellulitis, Nosocomial infections, Diarrhea, Meningitis in infants,Traveller's diarrhea, Hemorrhagic colitis, Hemolytic-uremic syndrome,Tularemia, Peptic ulcer, Gastric and Duodenal ulcers, Legionnaire'sDisease, Pontiac fever, Leptospirosis, Listeriosis, Leprosy (Hansen'sdisease), Tuberculosis, Gonorrhea, Ophthalmia neonatorum, Septicarthritis, Meningococcal disease including meningitis,Waterhouse-Friderichsen syndrome, Pseudomonas infection, Rocky mountainspotted fever, Typhoid fever type salmonellosis, Salmonellosis withgastroenteritis and enterocolitis, Bacillary dysentery/Shigellosis,Coagulase-positive staphylococcal infections: Localized skin infectionsincluding Diffuse skin infection (Impetigo), Deep localized infections,Acute infective endocarditis, Septicemia, Necrotizing pneumonia,Toxinoses such as Toxic shock syndrome and Staphylococcal foodpoisoning, Cystitis, Endometritis, Otitis media, Streptococcalpharyngitis, Scarlet fever, Rheumatic fever, Puerperal fever,Necrotizing fasciitis, Cholera, Plague (including Bubonic plague andPneumonic plague), as well as any infection caused by a bacteriaselected from but not limited to Helicobacter pyloris, Boreliaiburgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M.tuberculosis, M. avium, M. Intracellulare, M. kansaii, M gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter erogenes, Klebsiella pneuomiae, Pasteurellamulticoda, Bacteroides sp., Fusobacterium nucleatum, Sreptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomeyces israelli.

Non limiting examples of infectious disorder and/or disease caused byvirus is selected from the group consisting of but not limited toacquired immune deficiency (AIDS), West Nile encephalitis, coronavirusinfection, rhinovirus infection, influenza, dengue, hemorrhagic fever;an otological infection; severe acute respiratory syndrome (SARS), acutefebrile pharyngitis, pharyngoconjunctival fever, epidemickeratoconjunctivitis, infantile gastroenteritis, infectiousmononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis,hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1 infection,(gingivostomatitis in children, tonsillitis & pharyngitis in adults,keratoconjunctivitis), latent HSV-1 infection (herpes labialis, coldsores), aseptic meningitis, Cytomegalovirus infection, Cytomegalicinclusion disease, Kaposi sarcoma, Castleman disease, primary effusionlymphoma, influenza, measles, encephalitis, postinfectiousencephalomyelitis, Mumps, hyperplastic epithelial lesions (common, flat,plantar and anogenital warts, laryngeal papillomas, epidermodysplasiaverruciformis), croup, pneumonia, bronchiolitis, Poliomyelitis, Rabies,bronchiolitis, pneumonia, German measles, congenital rubella,Hemorrhagic Fever, Chickenpox, Dengue, Ebola infection, Echovirusinfection, EBV infection, Fifth Disease, Filovirus, Flavivirus, Hand,foot & mouth disease, Herpes Zoster Virus (Shingles), Human PapillomaVirus Associated Epidermal Lesions, Lassa Fever, Lymphocyticchoriomeningitis, Parainfluenza Virus Infection, Paramyxovirus,Parvovirus B19 Infection, Picornavirus, Poxviruses infection, Rotavirusdiarrhea, Rubella, Rubeola, Varicella, Variola infection.

An infectious disorder and/or disease caused by fungi optionallyincludes but is not limited to Allergic bronchopulmonary aspergillosis,Aspergilloma, Aspergillosis, Basidiobolomycosis, Blastomycosis,Candidiasis, Chronic pulmonary aspergillosis, Chytridiomycosis,Coccidioidomycosis, Conidiobolomycosis, Covered smut (barley),Cryptococcosis, Dermatophyte, Dermatophytid, Dermatophytosis, Endothrix,Entomopathogenic fungus, Epizootic lymphangitis, Epizootic ulcerativesyndrome, Esophageal candidiasis, Exothrix, Fungemia, Histoplasmosis,Lobomycosis, Massospora cicadina, Mycosis, Mycosphaerella fragariae,Myringomycosis, Paracoccidioidomycosis, Pathogenic fungi, Penicilliosis,Thousand cankers disease, Tinea, Zeaspora, Zygomycosis. Non limitingexamples of infectious disorder and/or disease caused by parasites isselected from the group consisting of but not limited to Acanthamoeba,Amoebiasis, Ascariasis, Ancylostomiasis, Anisakiasis, Babesiosis,Balantidiasis, Baylisascariasis, Blastocystosis, Candiru, Chagasdisease, Clonorchiasis, Cochliomyia, Coccidia, Chinese Liver FlukeCryptosporidiosis, Dientamoebiasis, Diphyllobothriasis, Dioctophymerenalis infection, Dracunculiasis, Echinococcosis, Elephantiasis,Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis,Gnathostomiasis, Hymenolepiasis, Halzoun Syndrome, Isosporiasis,Katayama fever, Leishmaniasis, lymphatic filariasis, Malaria,Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Primary amoebicmeningoencephalitis, Parasitic pneumonia, Paragonimiasis, Scabies,Schistosomiasis, Sleeping sickness, Strongyloidiasis, Sparganosis,Rhinosporidiosis, River blindness, Taeniasis (cause of Cysticercosis),Toxocariasis, Toxoplasmosis, Trichinosis, Trichomoniasis, Trichuriasis,Trypanosomiasis, Tapeworm infection.

A preferred example of infectious disease is a disease caused by any ofhepatitis B, hepatitis C, infectious mononucleosis, EBV,cytomegalovirus, AIDS, HIV-1, HIV-2, tuberculosis, malaria andschistosomiasis.

As used herein, the term “vaccine” refers to a biological preparationthat improves immunity to a particular disease, wherein the vaccineincludes an antigen, such as weakened or killed forms of pathogen, itstoxins or one of its surface proteins, against which immune responsesare elicited. A vaccine typically includes an adjuvant as immunepotentiator to stimulate the immune system. As used herein, the term“therapeutic vaccine” and/or “therapeutic vaccination” refers to avaccine used to treat ongoing disease, such as infectious disease orcancer.

As used herein, the term “adjuvant” refers to an agent used to stimulatethe immune system and increase the response to a vaccine, without havingany specific antigenic effect in itself.

As used herein, the term LY6G6F and/or LY6G6F protein(s) refers to anyone of the proteins set forth in SEQ ID NO:1, and/or variants thereof,and/or orthologs and/or fragments thereof, and/or nucleic acid sequencesencoding for same, that are differentially expressed in cancers asrecited herein and/or in infectious disorders as recited herein, and/orimmune related disorders as recited herein, and/or that play a role inthe etiology of cancers, and/or in infectious disorders, and/or immunerelated disorders.

According to preferred embodiments, a LY6G6F fragment comprises an aminoacid sequence of LY6G6F ectodomain, set forth in any one of SEQ ID NOs:2, 59, 81, 96, and/or variants thereof. According to preferredembodiments, a LY6G6F ortholog comprises any one of SEQ ID NOs:20, 29.According to preferred embodiments, a nucleic acid sequence encodingLY6G6F protein comprises SEQ ID NO:33, 57 or 182.

As used herein, the term VSIG10 and/or VSIG10 protein(s) refers to anyone of the proteins set forth in any one of SEQ ID NOs:3, 5, and/orvariants thereof, and/or orthologs and/or fragments thereof, and/ornucleic acid sequences encoding for same, that are differentiallyexpressed in cancers as recited herein and/or in infectious disorders asrecited herein, and/or immune related disorders as recited herein,and/or that play a role in the etiology of cancers and/or in infectiousdisorders, and/or immune related disorders.

According to preferred embodiments, a VSIG10 fragment comprises an aminoacid sequence of VSIG10 ectodomain, set forth in any one of SEQ ID NOs:4, 6, 60, 61, 82-93, 97-100, and/or variants thereof, and/or an aminoacid sequence comprising a VSIG10 variant (SEQ ID NO:5) unique edgeportion, demonstrated in FIG. 2A. According to preferred embodiments, aVSIG10 ortholog comprises any one of SEQ ID NOs: 19, 30. According topreferred embodiments, a nucleic acid sequence encoding VSIG10 proteincomprises any one of SEQ ID NOs: 34, 35, 36, 183, or 184.

As used herein, the term TMEM25 and/or TMEM25 protein(s) refers to anyone of the proteins set forth in any one of SEQ ID NOs: 7, 39, and/orvariants thereof, and/or orthologs and/or fragments thereof, and/ornucleic acid sequences encoding for same, that are differentiallyexpressed in cancers as recited herein and/or in infectious disorders asrecited herein, and/or immune related disorders as recited herein,and/or that play a role in the etiology of cancers and/or in infectiousdisorders, and/or immune related disorders.

According to preferred embodiments, a TMEM25 fragment comprises an aminoacid sequence of TMEM25 ectodomain, set forth in any one of SEQ ID NOs:8, 39, 94, 101 and/or variants thereof. According to preferredembodiments, a TMEM25 ortholog comprises a protein having a sequenceaccording to any of SEQ ID NO: 9, and/or 28. According to preferredembodiments, a nucleic acid sequence encoding TMEM25 protein comprisesany one of SEQ ID NOs:37 or 185.

As used herein, the term LSR and/or LSR protein(s) refers to any one ofthe proteins set forth in any one of SEQ ID NOs: 11, 13, 15-18, 143,and/or variants thereof, and/or orthologs and/or fragments thereof,and/or nucleic acid sequences encoding for same, that are differentiallyexpressed in cancers as recited herein and/or in infectious disorders asrecited herein, and/or immune related disorders as recited herein,and/or that play a role in the etiology of cancers and/or in infectiousdisorders, and/or immune related disorders.

According to preferred embodiments, a LSR fragment comprises an aminoacid sequence of LSR ectodomain, set forth in any one of SEQ ID NOs:10,12, 14, 22, 47-50, 95, 102, and/or variants thereof, and/or an aminoacid sequence comprising a LSR variant (SEQ ID NO:18) unique edgeportion, demonstrated in FIG. 2G. An example of LSR orthologs ispresented in any one of SEQ ID NOs: 21, 31, 32. According to preferredembodiments, a nucleic acid sequence encoding LSR protein comprises anyone of SEQ ID NOs: 40-46, 132, 155, 188, 186, 187, 145, 154.

Without wishing to be limited by a single hypothesis, each of theLY6G6F, VSIG10, TMEM25 and/or LSR proteins according to at least someembodiments of the present invention, was predicted to be an immunecostimulatory protein, e.g., a B7 protein family member that is involvedin B7 immune co-stimulation including for example T cell responseselicited against cancer cells and that elicit effects on immunity suchas triggering of autoimmune effects.

As used herein, the term the “soluble ectodomain (ECD)” or “ectodomain”or “soluble LY6G6F, VSIG10, TMEM25 and/or LSR protein(s)/molecule(s)” ofLY6G6F, VSIG10, TMEM25 and/or LSR means non-cell-surface-bound (i.e.circulating) LY6G6F, VSIG10, TMEM25 and/or LSR molecules or any portionthereof, including, but not limited to: LY6G6F, VSIG10, TMEM25 and/orLSR-Ig fusion proteins, wherein the extracellular domain of LY6G6F,VSIG10, TMEM25 and/or LSR is fused to an immunoglobulin (Ig) moietyrendering the fusion molecule soluble, or fragments and derivativesthereof, proteins with the extracellular domain of LY6G6F, VSIG10,TMEM25 and/or LSR fused or joined with a portion of a biologicallyactive or chemically active protein such as the papillomavirus E7 geneproduct, melanoma-associated antigen p97 or HIV env protein, orfragments and derivatives thereof; hybrid (chimeric) fusion proteinssuch as LY6G6F, VSIG10, TMEM25 and/or LSR-Ig, or fragments andderivatives thereof. Such fusion proteins are described in greaterdetail below.

“Soluble LY6G6F, VSIG10, TMEM25 and/or LSR protein(s)/molecule(s)” alsoinclude LY6G6F, VSIG10, TMEM25 and/or LSR molecules with thetransmembrane domain removed to render the protein soluble, or fragmentsand derivatives thereof; fragments, portions or derivatives thereof, andsoluble LY6G6F, VSIG10, TMEM25 and/or LSR mutant molecules. The solubleLY6G6F, VSIG10, TMEM25 and/or LSR molecules used in the methodsaccording to at least some embodiments of the invention may or may notinclude a signal (leader) peptide sequence.

Fragments of LY6G6F Polypeptides

The term the “soluble ectodomain (ECD)” or “ectodomain” or “soluble”form of LY6G6F refers also to the nucleic acid sequences encoding thecorresponding proteins of LY6G6F “soluble ectodomain (ECD)” or“ectodomain” or “soluble LY6G6F proteins/molecules”). Optionally, theLY6G6F ECD refers to any one of the polypeptide sequences below and/orlisted in Table A below, and/or or fragments or variants thereofpossessing at least 80% sequence identity, more preferably at least 90%sequence identity therewith and even more preferably at least 95, 96,97, 98 or 99% sequence identity therewith, and/or conjugates thereof,and/or polynucleotides encoding same:

SEQ ID NO: 2, amino acid residues 17-234 (not including signal peptide,up till transmembrane) (FIG. 1A):ADNMQAIYVALGEAVELPCPSPPTLHGDEHLSWFCSPAAGSFTTLVAQVQVGRPAPDPGKPGRESRLRLLGNYSLWLEGSKEEDAGRYWCAVLGQHHNYQNWRVYDVLVLKGSQLSARAADGSPCNVLLCSVVPSRRMDSVTWQEGKGPVRGRVQSFWGSEAALLLVCPGEGLSEPRSRRPRIIRCLMTHNKGVSFSLAA SIDASPALCAPSTGWDMP,and fragments and variants thereof possessing at least 80% sequenceidentity, more preferably at least 90% sequence identity therewith andeven more preferably at least 95, 96, 97, 98 or 99% sequence identitytherewith. SEQ ID NO:59 represents an example of the LY6G6F ECDincluding signal peptide.

TABLE A SEQ ID NO: Amino acid sequence Description 81ADNMQAIYVALGEAVELPCPSPPTLHGDEHLSWFCSPAAG LY6G6F_IgV_domain aa 17-122SFTTLVAQVQVGRPAPDPGKPGRESRLRLLGNYSLWLEGS of seq id: 1KEEDAGRYWCAVLGQHHNYQNWRVYD

Optionally, the fragment is of at least about 62, 63, 64, 65 and soforth amino acids of the extracellular domain of LY6G6F protein, setforth in SEQ ID NO: 1, up to 228 amino acids of the LY6G6F proteinextracellular domain, optionally including any integral value between 62and 228 amino acids in length. Preferably, the fragment is of at leastabout 62 and up to 82 amino acids of the LY6G6F protein extracellulardomain, optionally including any integral value between 62 and 82 aminoacids in length. Also preferably the fragment is of at least about 95 upto 115 amino acids of the LY6G6F protein extracellular domain,optionally including any integral value between 95 and 115 amino acidsin length. Also preferably the fragment is of at least about 208 up to228 amino acids of the LY6G6F protein extracellular domain, optionallyincluding any integral value between 208 and 228 amino acids in length.More preferably, the fragment is about 72 or 106 or 218 amino acids. TheLY6G6F fragment protein according to at least some embodiments of theinvention may or may not include a signal peptide sequence, and may ormay not include 1, 2, 3, 4, or 5 contiguous amino acids from the LY6G6Ftransmembrane domain.

In particular, the fragments of the extracellular domain of LY6G6F caninclude any sequence corresponding to any portion of or comprising theIgV domain of the extracellular domain of LY6G6F, having any sequencecorresponding to residues of LY6G6F (SEQ ID NO:1) starting from anyposition between 14 and 27 and ending at any position between 112 and132.

The LY6G6F proteins contain an immunoglobulin domain within theextracellular domain, the IgV domain (or V domain), shown in FIG. 1 A ina box, which is related to the variable domain of antibodies. The IgVdomain may be responsible for receptor binding, by analogy to the otherB7 family members. The Ig domain of the extracellular domain includesone disulfide bond formed between intradomain cystein residues, as istypical for this fold and may be important for structure-function. InSEQ ID NO: 1 these cysteines are located at residues 35 and 106.

In one embodiment, there is provided a soluble fragment of LY6G6F; asdescribed in greater detail below with regard to the section on fusionproteins, such a soluble fragment may optionally be described as a firstfusion partner. Useful fragments are those that retain the ability tobind to their natural receptor or receptors and/or retain the ability toinhibit T cell activation. A LY6G6F polypeptide that is a fragment offull-length LY6G6F typically has at least 20 percent, 30 percent, 40percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95percent, 98 percent, 99 percent, 100 percent, or even more than 100percent of the ability to bind its natural receptor(s) and/or of theability to inhibit T cell activation as compared to full-length LY6G6F.Soluble LY6G6F polypeptide fragments are fragments of LY6G6Fpolypeptides that may be shed, secreted or otherwise extracted from theproducing cells. In other embodiments, the soluble fragments of LY6G6Fpolypeptides include fragments of the LY6G6F extracellular domain thatretain LY6G6F biological activity, such as fragments that retain theability to bind to their natural receptor or receptors and/or retain theability to inhibit T cell activation. The extracellular domain caninclude 1, 2, 3, 4, or 5 contiguous amino acids from the transmembranedomain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signalsequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments the LY6G6F extracellular domain polypeptidecomprises the amino acid sequence of the IgV domain as set forth in anyone of SEQ ID NO: 81, or fragments or variants thereof, or the regionbetween the conserved cysteines of the IgV domain located at residues 35and 106 of the full-length protein SEQ ID NO:1, corresponding to thesequence set forth in SEQ ID NO: 96:CPSPPTLHGDEHLSWFCSPAAGSFTTLVAQVQVGRPAPDPGKPGRESRLRLLGNYSLWLEGSKEEDAGRYWC. In other embodiments the LY6G6F extracellular domainpolypeptide consists essentially of the amino acid sequence of the IgVdomain as set forth in any one of SEQ ID NOs: 81 and 96.

Generally, the LY6G6F polypeptide fragments are expressed from nucleicacids that include sequences that encode a signal sequence. The signalsequence is generally cleaved from the immature polypeptide to producethe mature polypeptide lacking the signal sequence. The signal sequenceof LY6G6F can be replaced by the signal sequence of another polypeptideusing standard molecule biology techniques to affect the expressionlevels, secretion, solubility, or other property of the polypeptide. Thesignal peptide sequence that is used to replace the LY6G6F signalpeptide sequence can be any known in the art.

Optionally, the LY6G6F ECD refers to any one of the nucleic acidsequences encoding LY6G6F ECD polypeptides, optionally to the nucleicacid sequences set forth in SEQ ID NO:33, or fragments thereof and/ordegenerative variants thereof, encoding LY6G6F ECD polypeptides setforth in SEQ ID NO:2.

Optionally, the LY6G6F ECD refers to orthologous ECD polypeptides.Optionally, the LY6G6F ECD refers to mouse LY6G6F ECD polypeptides, setforth in SEQ ID NOs:20, and/or a mouse LY6G6F ECD-IgG2a-Fc-fusedpolypeptide, set forth in SEQ ID NOs:23.

Fragments of VSIG10 Polypeptides

The term the “soluble ectodomain (ECD)” or “ectodomain” or “soluble”form of VSIG10 refers also to the nucleic acid sequences encoding thecorresponding proteins of VSIG10 “soluble ectodomain (ECD)” or“ectodomain” or “soluble VSIG10 proteins/molecules”). Optionally, theVSIG10 ECD refers to any one of the polypeptide sequences below and/orlisted in Table B below, and/or fragments or variants thereof possessingat least 80% sequence identity, more preferably at least 90% sequenceidentity therewith and even more preferably at least 95, 96, 97, 98 or99% sequence identity therewith, and/or conjugates thereof, and/orpolynucleotides encoding same:

SEQ ID NO: 4, amino acid residues 31-413 (not including signal peptide,up till transmembrane) (FIG. 1B):VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGIYTCQEILNVTQWFQVWLQVASGPYQIEVHIVATGTLPNGTLYAARGSQVDFSCNSSSRPPPVVEWWFQALNSSSESFGHNLTVNFFSLLLISPNLQGNYTCLALNQLSKRHRKVTTELLVYYPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLSVKEPLNIGG; SEQ ID NO: 6, amino acid residues31-312 (skipping exon 3 variant, not including signal peptide, up tilltransmembrane) (FIG. 1C):VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGIYTCQEILNVTQWFQVWLQVANPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLSVKEPLNIGG,and variants thereof possessing at least 80% sequence identity, morepreferably at least 90% sequence identity therewith and even morepreferably at least 95, 96, 97, 98 or 99% sequence identity therewith.SEQ ID NOs:60-61 represent examples of the VSIG10 ECD including signalpeptide.

TABLE B SEQ ID NO: Amino acid sequence Description 82VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEP VSIG10_IgC2_domain_1 aa 31-119VFLLSSNSSLRPAEPRFSLVDATSLHIESLSLG of seq id: 3 aa 31-119 ofDEGIYTCQEILNVTQWFQVWLQV 1 seq id: 5 83 PYQIEVHIVATGTLPNGTLYAARGSQVDFSCNSVSIG10_IgC2_domain_2 aa 123-215 SSRPPPVVEWWFQALNSSSESFGHNLTVNFFSL of seqid: 3 LLISPNLQGNYTCLALNQLSKRHRKVT 84 PPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDVSIG10_IgC2_domain_3 aa FLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKF 223-309 of seqid: 3 aa KCVTSHIVGPESGASCMVQIR 122-208 of seq id: 5 85PSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKIL VSIG10_IgC2_domain_4 aa 311-404WLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHN of seq id: 3 aa 210-303CSQDLDEGYYICRADSPVGVREMEIWLS of seq id: 5 86VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEP VSIG10_WT_IgC2_domains_1-2VFLLSSNSSLRPAEPRFSLVDATSLHIESLSLG aa 31-215 of seq id: 3DEGIYTCQEILNVTQWFQVWLQVASGPYQIEVH IVATGTLPNGTLYAARGSQVDFSCNSSSRPPPVVEWWFQALNSSSESFGHNLTVNFFSLLLISPNL QGNYTCLALNQLSKRHRKVT 87VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEP VSIG10_WT_IgC2_domains_1-3VFLLSSNSSLRPAEPRFSLVDATSLHIESLSLG aa 31-309 of seq id: 3DEGIYTCQEILNVTQWFQVWLQVASGPYQIEVH IVATGTLPNGTLYAARGSQVDFSCNSSSRPPPVVEWWFQALNSSSESFGHNLTVNFFSLLLISPNL QGNYTCLALNQLSKRHRKVTTELLVYYPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEE PGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIR 88 VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVSIG10_WT_IgC2_domains_1-4 VFLLSSNSSLRPAEPRFSLVDATSLHIESLSLG aa 31-404of seq id: 3 DEGIYTCQEILNVTQWFQVWLQVASGPYQIEVHIVATGTLPNGTLYAARGSQVDFSCNSSSRPPPV VEWWFQALNSSSESFGHNLTVNFFSLLLISPNLQGNYTCLALNQLSKRHRKVTTELLVYYPPPSAP QCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSH IVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSR HLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLS 89 PYQIEVHIVATGTLPNGTLYAARGSQVDFSCNSVSIG10_WT_IgC2_domains_2-3 SSRPPPVVEWWFQALNSSSESFGHNLTVNFFSL aa 123-309of seq LLISPNLQGNYTCLALNQLSKRHRKVTTELLVY id: 3YPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDP DFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIR 90 PYQIEVHIVATGTLPNGTLYAARGSQVDFSCNSVSIG10_WT_IgC2_domains_2-4 SSRPPPVVEWWFQALNSSSESFGHNLTVNFFSL aa 123-404of seq id: 3 LLISPNLQGNYTCLALNQLSKRHRKVTTELLVYYPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDP DFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKT CFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYY ICRADSPVGVREMEIWLS 91PPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPD VSIG10_IgC2_domains_3-4 aaFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKF 223-404 of seq id: 3 aa 122-303KCVTSHIVGPESGASCMVQIRGPSLLSEPMKTC of seq id: 5FTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVI IQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLS 92 VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVSIG10_Variant_skipping_exon_3_T95617_P6_IgC2_domains_1,VFLLSSNSSLRPAEPRFSLVDATSLHI 3 aa 31-208 of seq id: 5ESLSLGDEGIYTCQEILNVTQWFQVWLQVANPP PSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSD GKKFKCVTSHIVGPESGASCMVQIR 93VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVSIG10_Variant_skipping_exon_3_T95617_P6_IgC2_domains_1,VFLLSSNSSLRPAEPRFSLVDATSLHI 3-4 aa 31-303 of seq id: 5ESLSLGDEGIYTCQEILNVTQWFQVWLQVANPP PSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSD GKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILW LRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLS

Optionally, the fragment is of at least about 36, 37, 38, 39, 40, 41,42, 43, and so forth amino acids of the extracellular domain of VSIG10protein, set forth in SEQ ID NO:3, up to 393 amino acids of the VSIG10protein extracellular domain, optionally, including any integral valuebetween 36 and 393 amino acids in length. Preferably, the fragment is ofat least about 36 up to 70 amino acids of the VSIG10 proteinextracellular domain, optionally including any integral value between 36and 70 amino acids in length. Also preferably the fragment is of atleast about 80 up to 100 amino acids of the VSIG10 protein extracellulardomain, optionally including any integral value between 80 and 100 aminoacids in length. Also preferably the fragment is of at least about 170up to 200 amino acids of the VSIG10 protein extracellular domain,optionally including any integral value between 170 and 200 amino acidsin length. Also preferably the fragment is of at least about 265 up to290 amino acids of the VSIG10 protein extracellular domain, optionallyincluding any integral value between 265 and 290 amino acids in length.Also preferably the fragment is of at least about 365 up to 393 aminoacids of the VSIG10 protein extracellular domain, optionally includingany integral value between 365 and 393 amino acids in length. Morepreferably, the fragment is about 46, 49, 58, 60, 87, 89, 93, 94, 178,182, 185, 187, 273, 279, 282, 374, 383 amino acids. The VSIG10 fragmentprotein according to at least some embodiments of the invention may ormay not include a signal peptide sequence, and may or may not include 1,2, 3, 4, or 5 contiguous amino acids from the VSIG10 transmembranedomain.

In particular, the fragments of the extracellular domain of VSIG10 caninclude any sequence corresponding to any portion of or comprising ofone or more of the IgC2 domains of the extracellular domain of VSIG10,having any sequence corresponding to residues of VSIG10 (SEQ ID NO:3)starting from any position between 28 and 41 and ending at any positionbetween 109 and 122 or starting from any position between 120 and 133and ending at any position between 205 and 222 or starting from anyposition between 216 and 233 and ending at any position between 299 and310 or starting from any position between 310 and 321 and ending at anyposition between 394 and 414 or starting from any position between 28and 41 and ending at any position between 205 and 222 or starting fromany position between 28 and 41 and ending at any position between 299and 310 or starting from any position between 28 and 41 and ending atany position between 394 and 414 or starting from any position between120 and 133 and ending at any position between 299 and 310 or startingfrom any position between 120 and 133 and ending at any position between394 and 414 or starting from any position between 216 and 233 and endingat any position between 394 and 414, or having any sequencecorresponding to residues of VSIG10_Variant_skipping_exon_3_T95617_P6(SEQ ID NO:5) starting from any position between 28 and 41 and ending atany position between 198 and 209 or starting from any position between28 and 41 and ending at any position between 293 and 313.

The VSIG10 proteins contain immunoglobulin domains within theextracellular domain, IgC2 domain (or Ig-like C2 domain or Ig C2-setdomain), which is related to the constant domain of antibodies. Thedomains are illustrated in FIG. 1B (for SEQ ID NO:3) snd in FIG. 1C (forSEQ ID NO:5). The Ig domains of the extracellular domain include onedisulfide bond formed between intradomain cystein residues, as istypical for this fold and may be important for structure-function. InSEQ ID NO: 3 these cysteines are located at residues 44 and 103 and atresidues 153 and 201 and at residues 245 and 290 and at residues 331 and388. In SEQ ID NO:5 these cysteines are located at residues 44 and 103and 144 and 189 and at residues 230 and 287.

In one embodiment, there is provided a soluble fragment of VSIG10, whichmay optionally be described as a first fusion partner in the belowsection on fusion proteins. Useful fragments are those that retain theability to bind to their natural receptor or receptors and/or retain theability to inhibit T cell activation. A VSIG10 polypeptide that is afragment of full-length VSIG10 typically has at least 20 percent, 30percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90percent, 95 percent, 98 percent, 99 percent, 100 percent, or even morethan 100 percent of the ability to bind its natural receptor(s) and/orof the ability to inhibit T cell activation as compared to full-lengthVSIG10. Soluble VSIG10 polypeptide fragments are fragments of VSIG10polypeptides that may be shed, secreted or otherwise extracted from theproducing cells. In other embodiments, the soluble fragments of VSIG10polypeptides include fragments of the VSIG10 extracellular domain thatretain VSIG10 biological activity, such as fragments that retain theability to bind to their natural receptor or receptors and/or retain theability to inhibit T cell activation. The extracellular domain caninclude 1, 2, 3, 4, or 5 contiguous amino acids from the transmembranedomain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signalsequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments the VSIG10 extracellular domain polypeptidecomprises the amino acid sequence of at least one of the IgC2 domains asset forth in any one of SEQ IDS NO: 82, 83, 84 and 85, or fragments orvariants thereof, or the regions between the conserved cysteines of theIgC2 domains located at residues 44 and 103 of the full-length proteinSEQ ID NO:3, corresponding to the sequence set forth in SEQ ID NO: 97:CGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGI YTC, orlocated at residues 153 and 201 of the full-length protein SEQ ID NO:3,corresponding to the sequence set forth in SEQ ID NO: 98:CNSSSRPPPVVEWWFQALNSSSESFGHNLTVNFFSLLLISPNLQGNYTC or located at residues245 and 209 of the full-length protein SEQ ID NO:3, corresponding to thesequence set forth in SEQ ID NO: 99:CRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKC or located at residues331 and 388 of the full-length protein SEQ ID NO:3, corresponding to thesequence set forth in SEQ ID NO: 100:CQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYI C. In somefurther embodiments the VSIG10 extracellular domain polypeptide consistsessentially of amino acid sequence of at least one of SEQ IDS NOs:82-93, 97-100.

Generally, the VSIG10 polypeptide fragments are expressed from nucleicacids that include sequences that encode a signal sequence. The signalsequence is generally cleaved from the immature polypeptide to producethe mature polypeptide lacking the signal sequence. The signal sequenceof VSIG10 can be replaced by the signal sequence of another polypeptideusing standard molecule biology techniques to affect the expressionlevels, secretion, solubility, or other property of the polypeptide. Thesignal peptide sequence that is used to replace the VSIG10 signalpeptide sequence can be any known in the art.

Optionally, the VSIG10 ECD refers also to any one of the nucleic acidsequences encoding VSIG10 ECD polypeptides, optionally to the nucleicacid sequences set forth in SEQ ID NOs:34, 36, or fragments thereofand/or degenerative variants thereof, encoding VSIG10 ECD polypeptidesset forth in SEQ ID NOs:4, 6, respectively.

Optionally, the VSIG10 ECD refers to orthologous ECD polypeptides.Optionally, the VSIG10 ECD refers to mouse VSIG10 ECD polypeptides, setforth in SEQ ID NO:19, and/or a mouse VSIG10 ECD-IgG2a-Fc-fusedpolypeptide, set forth in SEQ ID NO:24.

Fragments of TMEM25 Polypeptides

The term the “soluble ectodomain (ECD)” or “ectodomain” or “soluble”form of TMEM25 refers also to the nucleic acid sequences encoding thecorresponding proteins of TMEM25 “soluble ectodomain (ECD)” or“ectodomain” or “soluble TMEM25 proteins/molecules”). Optionally, theTMEM25 ECD refers to any one of the polypeptide sequences below and/orlisted in Table C below, and/or fragments or variants thereof possessingat least 80% sequence identity, more preferably at least 90% sequenceidentity therewith and even more preferably at least 95, 96, 97, 98 or99% sequence identity therewith, and/or conjugates thereof, and/orpolynucleotides encoding same:

SEQ ID NO: 8, amino acid residues 27-232 (not including signal peptide,up till transmembrane) (FIG. ID):ELEPQIDGQTWAERALRENERHAFTCRVAGGPGTPRLAWYLDGQLQEASTSRLLSVGGEAFSGGTSTFTVTAHRAQHELNCSLQDPRSGRSANASVILNVQFKPEIAQVGAKYQEAQGPGLLVVLFALVRANPPANVTWIDQDGPVTVNTSDFLVLDAQNYPWLTNHTVQLQLRSLAHNLSVVATNDVGVTSASLPAPGL LATRVE,and variants thereof possessing at least 80% sequence identity, morepreferably at least 90% sequence identity therewith and even morepreferably at least 95, 96, 97, 98 or 99% sequence identity therewith.SEQ ID NO:39 represents example of the TMEM25 ECD including signalpeptide.

TABLE C SEQ ID NO: Amino acid sequence Description 94PQIDGQTWAERALRENERHAFTCRVAGGPGTPR TMEM25_IgC2_domain aa 30-123LAWYLDGQLQEASTSRLLSVGGEAFSGGTSTFT of seq id: 7VTAHRAQHELNCSLQDPRSGRSANASVI

Optionally, the fragment is of at least about 46, 47, 48, 49, 50, 51,52, and so forth amino acids of the extracellular domain of TMEM25protein, set forth in SEQ ID NO:7, up to 216 amino acids of the TMEM25protein extracellular domain, optionally including any integral valuebetween 46 and 216 amino acids in length. Preferably, the fragment is ofat least about 46 up to 66 amino acids of the TMEM25 proteinextracellular domain, optionally including any integral value between 46and 66 amino acids in length. Also preferably the fragment is of atleast about 84 up to 104 amino acids of the TMEM25 protein extracellulardomain, optionally including any integral value between 84 and 104 aminoacids in length. Also preferably the fragment is of at least about 196up to 216 amino acids of the TMEM25 protein extracellular domain,optionally including any integral value between 196 and 216 amino acidsin length. More preferably, the fragment is about 56 or 94 or 206 aminoacids. The TMEM25 fragment protein according to at least someembodiments of the invention may or may not include a signal peptidesequence, and may or may not include 1, 2, 3, 4, or 5 contiguous aminoacids from the TMEM25 transmembrane domain.

In particular, the fragments of the extracellular domain of TMEM25 caninclude any sequence corresponding to any portion of or comprising theIgC2 domain of the extracellular domain of TMEM25, having any sequencecorresponding to residues of TMEM25 (SEQ ID NO:7) starting from anyposition between 27 and 40 and ending at any position between 113 and133.

The TMEM25 proteins contain an immunoglobulin domain within theextracellular domain, IgC2 domain (or Ig-like C2 domain or Ig C2-setdomain), which is related to the constant domain of antibodies. Thedomain is shown in FIG. 1D in a box. The Ig domain of the extracellulardomain includes one disulfide bond formed between intradomain cysteinresidues, as is typical for this fold and may be important forstructure-function. In SEQ ID NO: 7 these cysteines are located atresidues 52 and 107.

In one embodiment, there is provided a soluble fragment of TMEM25, whichmay optionally be described as a first fusion partner, as for example inthe detailed section on fusion proteins below. Useful fragments arethose that retain the ability to bind to their natural receptor orreceptors and/or retain the ability to inhibit T cell activation. ATMEM25 polypeptide that is a fragment of full-length TMEM25 typicallyhas at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent,70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent,100 percent, or even more than 100 percent of the ability to bind itsnatural receptor(s) and/or of the ability to inhibit T cell activationas compared to full-length TMEM25. Soluble TMEM25 polypeptide fragmentsare fragments of TMEM25 polypeptides that may be shed, secreted orotherwise extracted from the producing cells. In other embodiments, thesoluble fragments of TMEM25 polypeptides include fragments of the TMEM25extracellular domain that retain TMEM25 biological activity, such asfragments that retain the ability to bind to their natural receptor orreceptors and/or retain the ability to inhibit T cell activation. Theextracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acidsfrom the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous aminoacids from the signal sequence. Alternatively, the extracellular domaincan have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus,N-terminus, or both.

In some embodiments the TMEM25 extracellular domain polypeptidecomprises the amino acid sequence of IgC2 domain, as set forth in anyone of SEQ ID NO: 94, or fragments or variants thereof, or the regionbetween the conserved cysteines of the IgC2 domain located at residues52 and 107 of the full-length protein SEQ ID NO:7, corresponding to thesequence set forth in SEQ ID NO: 101:CRVAGGPGTPRLAWYLDGQLQEASTSRLLSVGGEAFSGGTSTFTVTAHRAQHEL NC. In otherembodiments the TMEM25 extracellular domain polypeptide consistsessentially of the amino acid sequence of the IgC2 domain as set forthin any one of SEQ ID NOs: 94 and 101.

Generally, the TMEM25 polypeptide fragments are expressed from nucleicacids that include sequences that encode a signal sequence. The signalsequence is generally cleaved from the immature polypeptide to producethe mature polypeptide lacking the signal sequence. The signal sequenceof TMEM25 can be replaced by the signal sequence of another polypeptideusing standard molecule biology techniques to affect the expressionlevels, secretion, solubility, or other property of the polypeptide. Thesignal peptide sequence that is used to replace the TMEM25 signalpeptide sequence can be any known in the art.

Optionally, the TMEM25 ECD refers also to any one of the nucleic acidsequences encoding TMEM25 ECD polypeptides, optionally to the nucleicacid sequences set forth in SEQ ID NO:37, or fragments thereof and/ordegenerative variants thereof, encoding TMEM25 ECD polypeptides setforth in SEQ ID NO:8

Optionally, the TMEM25 ECD refers to orthologous ECD polypeptides.Optionally, the TMEM25 ECD refers to mouse TMEM25 ECD polypeptides, setforth in SEQ ID NOs:9, and/or a mouse TMEM25 ECD-IgG2a-Fc-fusedpolypeptide, set forth in SEQ ID NOs:25.

Fragments of LSR Polypeptides

The term the “soluble ectodomain (ECD)” or “ectodomain” or “soluble”form of LSR refers also to the nucleic acid sequences encoding thecorresponding proteins of LSR “soluble ectodomain (ECD)” or “ectodomain”or “soluble LSR proteins/molecules”). Optionally, the LSR ECD refers toany one of the polypeptide sequences below and/or listed in Table Dbelow, and/or fragments or variants thereof possessing at least 80%sequence identity, more preferably at least 90% sequence identitytherewith and even more preferably at least 95, 96, 97, 98 or 99%sequence identity therewith, and/or conjugates thereof, and/orpolynucleotides encoding same:

SEQ ID NO: 12, LSR isoform A ECD (not including signal peptide, up tilltransmembrane) amino acid residues 42-211 (FIG. 1E):IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVL GRTSGVAELLPGFQAGPIED;SEQ ID NO: 14, LSR isoform B ECD (not including signal peptide, up tilltransmembrane) amino acid residues 42-192 (FIG. 1F):IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVL D; SEQ ID NO: 47, LSRisoform C secreted variant amino acid residues 42-533 (FIG. 1G):IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLVYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV; SEQ ID NO: 48, LSR isoform Dsecreted variant amino acid residues 42-532 (FIG. 1H)IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLVYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV; SEQ ID NO: 49, LSR isoform Esecreted variant amino acid residues 42-493 (FIG. 1I):IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGMYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESL VV; SEQ ID NO: 50,LSR isoform F secreted variant amino acid residues 42-552 (FIG. 1J):IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLGRTSGVAELLPGFQAGPIEVYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKK NLALSRESLVV,

and variants thereof possessing at least 80% sequence identity, morepreferably at least 90% sequence identity therewith and even morepreferably at least 95, 96, 97, 98 or 99% sequence identity therewith.SEQ ID NOs:10, 22 represent example of the LSR ECD including signalpeptide.

Optionally, the fragment is of at least about 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110 and so forth amino acids of theextracellular domain of LSR protein, set forth in SEQ ID NO:11 and/or143, up to 198 amino acids of the extracellular domain, optionallyincluding any integral value between 100 and 198 amino acids in length.The LSR fragment protein according to at least some embodiments of theinvention may or may not include a signal peptide sequence, and may ormay not include 1, 2, 3, 4, or 5 contiguous amino acids from the LSRtransmembrane domain.

TABLE D SEQ ID NO: Amino acid sequence Description 95IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQP LSR_IgV_domain aa 42-186 ofIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAA seq id: 11, 13, 15, 16, 17, 18GNPGYNPYVECQDSVRTVRVVATKQGNAVTLGD YYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYA

Optionally, the fragment is of at least about 98, 99, 100, 101, 102 andso forth amino acids of the extracellular domain of LSR protein, setforth in SEQ ID NO: 11, up to 180 amino acids of the LY6G6F proteinextracellular domain, optionally including any integral value between 98and 180 amino acids in length Preferably, the fragment is of at leastabout 98 up to 118 amino acids of the LSR protein extracellular domain,optionally including any integral value between 98 and 118 amino acidsin length. Also preferably the fragment is of at least about 135 up to155 amino acids of the LSR protein extracellular domain, optionallyincluding any integral value between 135 and 155 amino acids in length.Also preferably the fragment is of at least about 160 up to 180 aminoacids of the LSR protein extracellular domain, optionally including anyintegral value between 160 and 180 amino acids in length. Morepreferably, the fragment is about 108 or 145 or 170 amino acids. The LSRfragment protein according to at least some embodiments of the inventionmay or may not include a signal peptide sequence, and may or may notinclude 1, 2, 3, 4, or 5 contiguous amino acids from the LSRtransmembrane domain.

The LSR proteins contain an immunoglobulin domain within theextracellular domain, the IgV domain (or V domain), which is related tothe variable domain of antibodies. The Ig domain is shown in a box inFIGS. 1E, 1F, 1G, 1H, and 1J, for SEQ ID NOs: 11, 13, 15, 16, and 18,respectively. The Ig domain of the extracellular domain includes onedisulfide bond formed between intradomain cystein residues, as istypical for this fold and may be important for structure-function. InSEQ ID NO: 11 these cysteines are located at residues 63 and 170.

In one embodiment, there is provided a soluble fragment of LSR, whichmay optionally be described as a first fusion partner, as for example inthe below section on fusion proteins. Useful fragments are those thatretain the ability to bind to their natural receptor or receptors and/orretain the ability to inhibit T cell activation. A LSR polypeptide thatis a fragment of full-length LSR typically has at least 20 percent, 30percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90percent, 95 percent, 98 percent, 99 percent, 100 percent, or even morethan 100 percent of the ability to bind its natural receptor(s) and/orof the ability to inhibit T cell activation as compared to full-lengthLSR. Soluble LSR polypeptide fragments are fragments of LSR polypeptidesthat may be shed, secreted or otherwise extracted from the producingcells. In other embodiments, the soluble fragments of LSR polypeptidesinclude fragments of the LSR extracellular domain that retain LSRbiological activity, such as fragments that retain the ability to bindto their natural receptor or receptors and/or retain the ability toinhibit T cell activation. The extracellular domain can include 1, 2, 3,4, or 5 contiguous amino acids from the transmembrane domain, and/or 1,2, 3, 4, or 5 contiguous amino acids from the signal sequence.Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or moreamino acids removed from the C-terminus, N-terminus, or both.

In some embodiments the LSR extracellular domain polypeptide comprisesthe amino acid of the IgV domain as set forth in any one of SEQ ID NO:95, or fragments or variants thereof, or the region between theconserved cysteines of the IgV domain located at residues 63 and 170 ofthe full-length protein SEQ ID NO:11, corresponding to the sequence setforth in SEQ ID NO: 102:CTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYC. In some furtherembodiments the LSR extracellular domain polypeptide consistsessentially of the amino acid of the IgV domain as set forth in any oneof SEQ ID NO: 95, and SEQ ID NO: 102.

Generally, the LSR polypeptide fragments are expressed from nucleicacids that include sequences that encode a signal sequence. The signalsequence is generally cleaved from the immature polypeptide to producethe mature polypeptide lacking the signal sequence. The signal sequenceof LSR can be replaced by the signal sequence of another polypeptideusing standard molecule biology techniques to affect the expressionlevels, secretion, solubility, or other property of the polypeptide. Thesignal peptide sequence that is used to replace the LSR signal peptidesequence can be any known in the art.

Optionally, the LSR ECD refers also to any one of the nucleic acidsequences encoding LSR ECD polypeptides, optionally to the nucleic acidsequences set forth in SEQ ID NO:40, 41, 132, 44, 155, 188, or fragmentsthereof and/or degenerative variants thereof, encoding LSR ECDpolypeptides set forth in any one of SEQ ID NO:12, 14, 47, 48, 49, 50,respectively.

Optionally, the LSR ECD refers to orthologous ECD polypeptides.Optionally, the LSR ECD refers to mouse LSR ECD polypeptides, set forthin SEQ ID NOs:21, and/or a mouse LSR ECD-IgG2a-Fc-fused polypeptide, setforth in SEQ ID NOs:26.

Variants of LY6G6F, VSIG10, TMEM25 and/or LSR Polypeptides

The present invention encompasses useful variants of LY6G6F, VSIG10,TMEM25 and/or LSR polypeptides including those that increase biologicalactivity, as indicated by any of the assays described herein, or thatincrease half life or stability of the protein. Soluble LY6G6F, VSIG10,TMEM25 and/or LSR proteins or fragments, or fusions thereof havingLY6G6F, VSIG10, TMEM25 and/or LSR proteins activity, respectively, canbe engineered to increase biological activity. In a further embodiment,the LY6G6F, VSIG10, TMEM25 and/or LSR proteins or fusion protein ismodified with at least one amino acid substitution, deletion, orinsertion that increases the binding of the molecule to an immune cell,for example a T cell, and transmits an inhibitory signal into the Tcell.

Other optional variants are those LY6G6F, VSIG10, TMEM25 and/or LSRproteins that are engineered to selectively bind to one type of T cellversus other immune cells. For example, the LY6G6F, VSIG10, TMEM25and/or LSR polypeptide can be engineered to bind optionally to Tregs,Th0, Th1, Th17, Th2 or Th22 cells. Preferential binding refers tobinding that is at least 10%, 20%, 30%, 40%, 50%, 60% f 70%, 80%, 90%,95%, or greater for one type of cell over another type of cell. Stillother variants of LY6G6F, VSIG10, TMEM25 and/or LSR protein can beengineered to have reduced binding to immune cells relative to wildtypeLY6G6F, VSIG10, TMEM25 and/or LSR protein, respectively. These variantscan be used in combination with variants having stronger bindingproperties to modulate the immune response with a moderate impact.

Also optionally, variant LY6G6F, VSIG10, TMEM25 and/or LSR protein canbe engineered to have an increased half-life relative to wildtype. Thesevariants typically are modified to resist enzymatic degradation.Exemplary modifications include modified amino acid residues andmodified peptide bonds that resist enzymatic degradation. Variousmodifications to achieve this are known in the art.

The LY6G6F protein (SEQ ID NO:1) also has the following non-silent SNPs(Single Nucleotide Polymorphism) as listed in Table E, (given accordingto their position(s) on the amino acid sequence, with the alternativeamino acid listed the presence of SNPs in LY6G6F protein (SEQ ID NO:1)sequence provides support for alternative sequence(s) of this proteinaccording to the present invention. SEQ ID NO:58 is an example of such aalternative sequence, with alternative amino-acids, using part of theSNPs below

TABLE E Amino acid mutations SNP position(s) on amino Alternative acidsequence amino acid(s) 34 P −> Q 39 P −> S 107 A −> T 167 R −> K

The LSR protein (SEQ ID NO:11) also has the following non-silent SNPs(Single Nucleotide Polymorphism) as listed in Table F, (given accordingto their position(s) on the amino acid sequence, with the alternativeamino acid listed; the presence of SNPs in LSR protein (SEQ ID NO:11)sequence provides support for alternative sequence(s) of this proteinaccording to the present invention. SEQ ID NO:143 is an example of sucha alternative sequence, with alternative amino-acids, using part of theSNPs below

TABLE F Amino acid mutations SNP position(s) on amino Alternative acidsequence amino acid(s) 209 I −> M 211 D −> G 260 L −> R 315 S −> N 382 A−> G 591 N −> D

The VSIG10 protein (SEQ ID NO:3) also has the following non-silent SNPs(Single Nucleotide Polymorphism) as listed in Table G, (given accordingto their position(s) on the amino acid sequence, with the alternativeamino acid listed; the presence of SNPs in VSIG10 protein (SEQ ID NO:3)sequence provides support for alternative sequence(s) of this proteinaccording to the present invention.

TABLE G Amino acid mutations SNP position(s) on amino Alternative acidsequence amino acid(s) 333 V −> M 435 H −> Y

The TMEM25 protein (SEQ ID NO:7) also has the following non-silent SNPs(Single Nucleotide Polymorphism) as listed in Table H, (given accordingto their position(s) on the amino acid sequence, with the alternativeamino acid listed; the presence of SNPs in TMEM25 protein (SEQ ID NO:7)sequence provides support for alternative sequence(s) of this proteinaccording to the present invention.

TABLE H Amino acid mutations SNP position(s) on amino Alternative acidsequence amino acid(s) 25 W −> C 342 Q −> R

Various aspects of the invention are described in further detail in thefollowing subsections.

Nucleic Acids

A “nucleic acid fragment” or an “oligonucleotide” or a “polynucleotide”are used herein interchangeably to refer to a polymer of nucleic acidresidues. A polynucleotide sequence of the present invention refers to asingle or double stranded nucleic acid sequences which is isolated andprovided in the form of an RNA sequence, a complementary polynucleotidesequence (cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

Thus, the present invention encompasses nucleic acid sequences describedhereinabove; fragments thereof, sequences hybridizable therewith,sequences homologous thereto [e.g., at least 90%, at least 95, 96, 97,98 or 99% or more identical to the nucleic acid sequences set forthherein], sequences encoding similar polypeptides with different codonusage, altered sequences characterized by mutations, such as deletion,insertion or substitution of one or more nucleotides, either naturallyoccurring or man induced, either randomly or in a targeted fashion. Thepresent invention also encompasses homologous nucleic acid sequences(i.e., which form a part of a polynucleotide sequence of the presentinvention), which include sequence regions unique to the polynucleotidesof the present invention.

Thus, the present invention also encompasses polypeptides encoded by thepolynucleotide sequences of the present invention. The present inventionalso encompasses homologues of these polypeptides, such homologues canbe at least 90%, at least 95, 96, 97, 98 or 99% or more homologous tothe amino acid sequences set forth below, as can be determined usingBlastP software of the National Center of Biotechnology Information(NCBI) using default parameters. As mentioned hereinabove, biomolecularsequences of the present invention can be efficiently utilized as tissueor pathological markers and as putative drugs or drug targets fortreating or preventing a disease.

Oligonucleotides designed for carrying out the methods of the presentinvention for any of the sequences provided herein (designed asdescribed above) can be generated according to any oligonucleotidesynthesis method known in the art such as enzymatic synthesis or solidphase synthesis. Oligonucleotides used according to this aspect of thepresent invention are those having a length selected from a range ofabout 10 to about 200 bases preferably about 15 to about 150 bases, morepreferably about 20 to about 100 bases, most preferably about 20 toabout 50 bases.

The oligonucleotides of the present invention may comprise heterocyclicnucleosides consisting of purines and the pyrimidines bases, bonded in a3′ to 5′ phosphodiester linkage.

Preferable oligonucleotides are those modified in either backbone,internucleoside linkages or bases, as is broadly described hereinunder.Such modifications can oftentimes facilitate oligonucleotide uptake andresistivity to intracellular conditions.

Specific examples of preferred oligonucleotides useful according to thisaspect of the present invention include oligonucleotides containingmodified backbones or non-natural internucleoside linkages.Oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466, 677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andamino alkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms can also be used.

Alternatively, modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; and 5,677,439.

Other oligonucleotides which can be used according to the presentinvention, are those modified in both sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example for such anoligonucleotide mimetic, includes peptide nucleic acid (PNA). A PNAoligonucleotide refers to an oligonucleotide where the sugar-backbone isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The bases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Other backbone modifications, which can be used in thepresent invention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides of the present invention may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified bases include but are not limited to other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further bases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science andEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Such bases areparticularly useful for increasing the binding affinity of theoligomeric compounds according to at least some embodiments of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.[Sanghvi Y S et al. (1993) Antisense Research and Applications, CRCPress, Boca Raton 276-278] and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides according to at least someembodiments of the invention involves chemically linking to theoligonucleotide one or more moieties or conjugates, which enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, asdisclosed in U.S. Pat. No. 6,303,374.

It is not necessary for all positions in a given oligonucleotidemolecule to be uniformly modified, and in fact more than one of theaforementioned modifications may be incorporated in a single compound oreven at a single nucleoside within an oligonucleotide.

Peptides

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

Polypeptide products can be biochemically synthesized such as byemploying standard solid phase techniques. Such methods includeexclusive solid phase synthesis, partial solid phase synthesis methods,fragment condensation, classical solution synthesis. These methods arepreferably used when the peptide is relatively short (i.e., 10 kDa)and/or when it cannot be produced by recombinant techniques (i.e., notencoded by a nucleic acid sequence) and therefore involves differentchemistry.

Solid phase polypeptide synthesis procedures are well known in the artand further described by John Morrow Stewart and Janis Dillaha Young,Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic polypeptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

In cases where large amounts of a polypeptide are desired, it can begenerated using recombinant techniques such as described by Bitter etal., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990)Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514,Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565.

It will be appreciated that peptides identified according to theteachings of the present invention may be degradation products,synthetic peptides or recombinant peptides as well as peptidomimetics,typically, synthetic peptides and peptoids and semipeptoids which arepeptide analogs, which may have, for example, modifications renderingthe peptides more stable while in a body or more capable of penetratinginto cells. Such modifications include, but are not limited to Nterminus modification, C terminus modification, peptide bondmodification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O,O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted bysynthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Since the peptides of the present invention are preferably utilized intherapeutics which require the peptides to be in soluble form, thepeptides of the present invention preferably include one or morenon-natural or natural polar amino acids, including but not limited toserine and threonine which are capable of increasing peptide solubilitydue to their hydroxyl-containing side chain.

Expression Systems

To enable cellular expression of the polynucleotides of the presentinvention, a nucleic acid construct according to the present inventionmay be used, which includes at least a coding region of one of the abovenucleic acid sequences, and further includes at least one cis actingregulatory element. As used herein, the phrase “cis acting regulatoryelement” refers to a polynucleotide sequence, preferably a promoter,which binds a trans acting regulator and regulates the transcription ofa coding sequence located downstream thereto.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention.

Preferably, the promoter utilized by the nucleic acid construct of thepresent invention is active in the specific cell population transformed.Examples of cell type-specific and/or tissue-specific promoters includepromoters such as albumin that is liver specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). The nucleic acid construct of the presentinvention can further include an enhancer, which can be adjacent ordistant to the promoter sequence and can function in up regulating thetranscription therefrom.

The nucleic acid construct of the present invention preferably furtherincludes an appropriate selectable marker and/or an origin ofreplication. Preferably, the nucleic acid construct utilized is ashuttle vector, which can propagate both in E. coli (wherein theconstruct comprises an appropriate selectable marker and origin ofreplication) and be compatible for propagation in cells, or integrationin a gene and a tissue of choice. The construct according to the presentinvention can be, for example, a plasmid, a bacmid, a phagemid, acosmid, a phage, a virus or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3,pcDNA3.1 (+/−), pGL3, PzeoSV2 (+/−), pDisplay, pEF/myc/cyto,pCMV/myc/cyto each of which is commercially available from InvitrogenCo. (www.invitrogen.com). Examples of retroviral vector and packagingsystems are those sold by Clontech, San Diego, Calif., including Retro-Xvectors pLNCX and pLXSN, which permit cloning into multiple cloningsites and the transgene is transcribed from CMV promoter. Vectorsderived from Mo-MuLV are also included such as pBabe, where thetransgene will be transcribed from the 5′ LTR promoter.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. The most preferredconstructs for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral construct suchas a retroviral construct includes at least one transcriptionalpromoter/enhancer or locus-defining elements, or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used, unless it is alreadypresent in the viral construct. In addition, such a construct typicallyincludes a signal sequence for secretion of the peptide from a host cellin which it is placed. Preferably the signal sequence for this purposeis a mammalian signal sequence or the signal sequence of thepolypeptides of the present invention. Optionally, the construct mayalso include a signal that directs polyadenylation, as well as one ormore restriction sites and a translation termination sequence. By way ofexample, such constructs will typically include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second-strand DNA synthesis, anda 3′ LTR or a portion thereof. Other vectors can be used that arenon-viral, such as cationic lipids, polylysine, and dendrimers.

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a proteinaccording to at least some embodiments of the invention, or derivatives,fragments, analogs or homologs thereof. As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. Examples of vectortypes are plasmids and viral vectors. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors”. The invention isintended to include such forms of expression vectors, such as plasmids,viral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors according to at least someembodiments of the invention comprise a nucleic acid according to atleast some embodiments of the invention in a form suitable forexpression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, thatis operatively-linked to the nucleic acid sequence to be expressed.Within a recombinant expression vector, “operably-linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequences in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell).

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. The expression vectors according to at least some embodiments ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

The recombinant expression vectors according to at least someembodiments of the invention can be designed for production of variantproteins in prokaryotic or eukaryotic cells. For example, proteinsaccording to at least some embodiments of the invention can be expressedin bacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, to the amino or C terminus of the recombinant protein.Such fusion vectors typically serve three purposes: (i) to increaseexpression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin,PreScission, TEV and enterokinase. Typical fusion expression vectorsinclude pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia,Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

In another embodiment, the expression vector encoding for the protein ofthe invention is a yeast expression vector. Examples of vectors forexpression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari,et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982.Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123),pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogenCorp, San Diego, Calif.).

Alternatively, polypeptides of the present invention can be produced ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840)and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195), pIRESpuro(Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen) pREP4 (Invitrogen),pcDNA3 (Invitrogen). When used in mammalian cells, the expressionvectors control functions are often provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,adenovirus 2, cytomegalovirus, Rous Sarcoma Virus, and simian virus 40.For other suitable expression systems for both prokaryotic andeukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the alpha-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

According to at least some embodiments the invention further provides arecombinant expression vector comprising a DNA molecule according to atleast some embodiments of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively-linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to mRNA encoding for protein according to at leastsome embodiments of the invention. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see, e.g.,Weintraub, et al., “Antisense RNA as a molecular tool for geneticanalysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.

According to at least some embodiments the invention pertains to hostcells into which a recombinant expression vector according to at leastsome embodiments of the invention has been introduced. The terms “hostcell” and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut also to the progeny or potential progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,protein according to at least some embodiments of the invention can beproduced in bacterial cells such as E. coli, insect cells, yeast, plantor mammalian cells (such as Chinese hamster ovary cells (CHO) or COS or293 cells). Other suitable host cells are known to those skilled in theart.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin, puromycin, blasticidin and methotrexate. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding protein according to at least someembodiments of the invention or can be introduced on a separate vector.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell according to at least some embodiments of the invention,such as a prokaryotic or eukaryotic host cell in culture, can be used toproduce (i.e., express) protein according to at least some embodimentsof the invention. Accordingly, the invention further provides methodsfor producing proteins according to at least some embodiments of theinvention using the host cells according to at least some embodiments ofthe invention. In one embodiment, the method comprises culturing thehost cell of the present invention (into which a recombinant expressionvector encoding protein according to at least some embodiments of theinvention has been introduced) in a suitable medium such that theprotein according to at least some embodiments of the invention isproduced. In another embodiment, the method further comprises isolatingprotein according to at least some embodiments of the invention from themedium or the host cell.

For efficient production of the protein, it is preferable to place thenucleotide sequences encoding the protein according to at least someembodiments of the invention under the control of expression controlsequences optimized for expression in a desired host. For example, thesequences may include optimized transcriptional and/or translationalregulatory sequences (such as altered Kozak sequences).

It should be noted, that according to at least some embodiments of thepresent invention the LY6G6F, VSIG10, TMEM25 and/or LSR proteinsaccording to at least some embodiments of the invention may be isolatedas naturally-occurring polypeptides, or from any source whether natural,synthetic, semi-synthetic or recombinant. Accordingly, the LY6G6F,VSIG10, TMEM25 and/or LSR proteins may be isolated asnaturally-occurring proteins from any species, particularly mammalian,including bovine, ovine, porcine, murine, equine, and preferably human.Alternatively, the LY6G6F, VSIG10, TMEM25 and/or LSR proteins may beisolated as recombinant polypeptides that are expressed in prokaryote oreukaryote host cells, or isolated as a chemically synthesizedpolypeptide.

A skilled artisan can readily employ standard isolation methods toobtain isolated LY6G6F, VSIG10, TMEM25 and/or LSR proteins. The natureand degree of isolation will depend on the source and the intended useof the isolated molecules.

Transgenic Animals and Plants

According to at least some embodiments the invention also providestransgenic non-human animals and transgenic plants comprising one ormore nucleic acid molecules according to at least some embodiments ofthe invention that may be used to produce the polypeptides according toat least some embodiments of the invention. The polypeptides can beproduced in and recovered from tissue or bodily fluids, such as milk,blood or urine, of goats, cows, horses, pigs, rats, mice, rabbits,hamsters or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690,5,756,687, 5,750,172, and 5,741,957.

Non-human transgenic animals and transgenic plants are produced byintroducing one or more nucleic acid molecules according to at leastsome embodiments of the invention into the animal or plant by standardtransgenic techniques. The transgenic cells used for making thetransgenic animal can be embryonic stem cells, somatic cells orfertilized egg cells. The transgenic non-human organisms can bechimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See,e.g., Hogan et al. Manipulating the Mouse Embryo: A Laboratory Manual2ed. Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics andTransgenics: A Practical Approach, Oxford University Press (2000); andPinkert, Transgenic Animal Technology: A Laboratory Handbook, AcademicPress (1999).

Gene Therapy

According to at least some embodiments of the present invention, nucleicacid sequences encoding soluble LY6G6F, VSIG10, TMEM25 and/or LSRproteins can be used in gene therapy for treatment of infectiousdisorders, and/or immune related disorders, and or cancer.

As used herein, “gene therapy” is a process to treat a disease bygenetic manipulation so that a sequence of nucleic acid is transferredinto a cell, the cell then expressing any genetic product encoded by thenucleic acid. For example, as is well known by those skilled in the art,nucleic acid transfer may be performed by inserting an expression vectorcontaining the nucleic acid of interest into cells ex vivo or in vitroby a variety of methods including, for example, calcium phosphateprecipitation, diethyaminoethyl dextran, polyethylene glycol (PEG),electroporation, direct injection, lipofection or viral infection(Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press 1989); Kriegler M. Gene Transfer ad Expression:A Laboratory Manual (W. H. Freeman and Co, New York, N.Y., 1993) and Wu,Methods in Enzymology (Academic Press, New York, 1993). Alternatively,nucleic acid sequences of interest may be transferred into a cell invivo in a variety of vectors and by a variety of methods including, forexample, direct administration of the nucleic acid into a subject, orinsertion of the nucleic acid into a viral vector and infection of thesubject with the virus. Other methods used for in vivo transfer includeencapsulation of the nucleic acid into liposomes, and direct transfer ofthe liposomes, or liposomes combined with a hemagglutinating Sendaivirus, to a subject. The transfected or infected cells express theprotein products encoded by the nucleic acid in order to ameliorate adisease or the symptoms of a disease.

Antibodies and Immune System Response

As used herein, the terms “immunologic”, “immunological” or “immune”response is the development of a beneficial humoral (antibody mediated)and/or a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against a peptide in a recipientpatient. Such a response can be an active response induced byadministration of immunogen or a passive response induced byadministration of antibody or primed T-cells. Without wishing to belimited by a single hypothesis, a cellular immune response is elicitedby the presentation of polypeptide epitopes in association with Class Ior Class II MHC molecules to activate antigen-specific CD4+ T helpercells and/or CD8+ cytotoxic T cells. The response may also involveactivation of monocytes, macrophages, NK cells, basophils, dendriticcells, astrocytes, microglia cells, eosinophils, activation orrecruitment of neutrophils or other components of innate immunity. Thepresence of a cell-mediated immunological response can be determined byproliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte)assays. The relative contributions of humoral and cellular responses tothe protective or therapeutic effect of an immunogen can bedistinguished by separately isolating antibodies and T-cells from animmunized syngeneic animal and measuring protective or therapeuticeffect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

A “signal, transduction pathway” refers to the biochemical relationshipbetween varieties of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell.

As used herein, the phrase “cell surface receptor” includes, forexample, molecules and complexes of molecules capable of receiving asignal and the transmission of such a signal across the plasma membraneof a cell.

The term “antibody” as referred to herein includes whole polyclonal andmonoclonal antibodies and any antigen binding fragment (i.e.,“antigen-binding portion”) or single chains thereof. An “antibody”refers to a glycoprotein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, or an antigenbinding portion thereof. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:1-R1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., LY6G6F, VSIG10, TMEM25 and/or LSR molecules, and/or a fragmentthereof). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V Light, V Heavy, Constant light(CL) and CH1 domains; (ii) a F(ab′).2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds LY6G6F, VSIG10, TMEM25 or LSR proteins and/or fragments thereof,and is substantially free of antibodies that specifically bind antigensother than LY6G6F, VSIG10, TMEM25 or LSR, respectively. An isolatedantibody that specifically binds LY6G6F, VSIG10, TMEM25 or LSR proteinsmay, however, have cross-reactivity to other antigens, such as LY6G6F,VSIG10, TMEM25 or LSR molecules from other species, respectively.Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies according to at least some embodiments of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

As used herein, an antibody that “specifically binds to human LY6G6F,VSIG10, TMEM25 or LSR proteins” is intended to refer to an antibody thatbinds to LY6G6F, VSIG10, TMEM25 or LSR proteins, respectively, such asfor example, one with a KD of 5×10-8 M, 3×10-8 M, 1×.10-9 M or less.

The term “K-assoc” or “Ka”, as used herein, is intended to refer to theassociation rate of a particular antibody-antigen interaction, whereasthe term “Kdiss” or “Kd,” as used herein, is intended to refer to thedissociation rate of a particular antibody-antigen interaction. The term“KD”, as used herein, is intended to refer to the dissociation constant,which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and isexpressed as a molar concentration (M). KD values for antibodies can bedetermined using methods well established in the art. A preferred methodfor determining the KD of an antibody is by using surface Plasmonresonance, preferably using a biosensor system such as a Biacore®.system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a KD of 10-8 M or less, more preferably 10-9 M orless and even more preferably 10-10 M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a KD of 10-7 M or less, more preferably 10-8 M or less.

As used herein, the term “subject” or “patient” includes any human ornonhuman animal. The term “nonhuman animal” includes all vertebrates,e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs,cats, horses, cows, chickens, amphibians, reptiles, etc.

Anti-LY6G6F, Anti-VSIG10, Anti-TMEM25 and Anti-LSR Antibodies

The antibodies according to at least some embodiments of the inventionincluding those having the particular germline sequences, homologousantibodies, antibodies with conservative modifications, engineered andmodified antibodies are characterized by particular functional featuresor properties of the antibodies. For example, the antibodies bindspecifically to human LY6G6F, VSIG10, TMEM25 or LSR. Preferably, anantibody according to at least some embodiments of the invention bindsto corresponding LY6G6F, VSIG10, TMEM25 or LSR with high affinity, forexample with a KD of 10-8 M or less or 10-9 M or less or even 10-10 M orless. The anti-LY6G6F, anti-VSIG10, anti-TMEM25 and anti-LSR antibodiesaccording to at least some embodiments of the present inventionpreferably exhibit one or more of the following characteristics:

(i) binds to corresponding human LY6G6F, VSIG10, TMEM25 or LSR with a KDof 5.×10-8 M or less;

(ii) modulates (enhances or inhibits) B7 immune costimulation andrelated activities and functions such a T cell responses involved inantitumor immunity and autoimmunity, and/or

(iii) binds to LY6G6F, VSIG10, TMEM25 or LSR antigen expressed by cancercells including for example melanoma, cancers of liver, renal, brain,breast, colon, lung, ovary, pancreas, prostate, stomach, multiplemyeloma, and hematopoietic cancer, including but not limited to lymphoma(Hodgkin's and non Hodgkin's), acute and chronic lymphoblastic leukemiaand acute and chronic myeloid leukemia, but does not substantially bindto normal cells. In addition, preferably these antibodies and conjugatesthereof will be effective in eliciting selective killing of such cancercells and for modulating immune responses involved in autoimmunity andcancer.

More preferably, the antibody binds to corresponding human LY6G6F,VSIG10, TMEM25 or LSR antigen with a KD of 3×10-8 M or less, or with aKD of 1×10-9 M or less, or with a KD of 0.1.×10-9 M or less, or with aKD Of 0.05.×10-9 M or less or with a KD of between 1×10-9 and 1×10-11 M.

Standard assays to evaluate the binding ability of the antibodies towardLY6G6F, VSIG10, TMEM25 or LSR are known in the art, including forexample, ELISAs, Western blots and RIAs. Suitable assays are describedin detail in the Examples. The binding kinetics (e.g., binding affinity)of the antibodies also can be assessed by standard assays known in theart, such as by Biacore analysis.

Upon production of anti-LY6G6F, anti-VSIG10, anti-TMEM25 and anti-LSRantibody sequences from antibodies can bind to LY6G6F, VSIG10, TMEM25 orLSR the VH and VL sequences can be “mixed and matched” to create otheranti-LY6G6F, anti-VSIG10, anti-TMEM25 and anti-LSR, binding moleculesaccording to at least some embodiments of the invention. LY6G6F, VSIG10,TMEM25 or LSR binding of such “mixed and matched” antibodies can betested using the binding assays described above. e.g., ELISAs).Preferably, when VH and VL chains are mixed and matched, a VH sequencefrom a particular VH/VL pairing is replaced with a structurally similarVH sequence. Likewise, preferably a VL sequence from a particular VH/VLpairing is replaced with a structurally similar VL sequence. Forexample, the VH and VL sequences of homologous antibodies areparticularly amenable for mixing and matching.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of the invention comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody.

A human antibody that is “the product of” or “derived from” a particularhuman germline immunoglobulin sequence may contain amino aciddifferences as compared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a selected human antibody typically isat least 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the human antibody as being human when comparedto the germline immunoglobulin amino acid sequences of other species(e.g., murine germline sequences). In certain cases, a human antibodymay be at least 95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%,or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention comprises heavyand light chain variable regions comprising amino acid sequences thatare homologous to isolated anti-LY6G6F, anti-VSIG10, anti-TMEM25 oranti-LSR amino acid sequences of preferred anti-LY6G6F, anti-VSIG10,anti-TMEM25 or anti-LSR antibodies, respectively, wherein the antibodiesretain the desired functional properties of the parent anti-LY6G6F,anti-VSIG10, anti-TMEM25 or anti-LSR antibodies.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availablecommercially), using either a Blossum 62 matrix or a PAM250 matrix, anda gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody moleculesaccording to at least some embodiments of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on preferred anti-LY6G6F, anti-VSIG10, anti-TMEM25 oranti-LSR antibodies isolated and produced using methods herein, orconservative modifications thereof, and wherein the antibodies retainthe desired functional properties of the anti-LY6G6F, anti-VSIG10,anti-TMEM25 or anti-LSR antibodies according to at least someembodiments of the invention, respectively.

In various embodiments, the anti-LY6G6F, anti-VSIG10, anti-TMEM25 oranti-LSR antibody can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody according to at least some embodiments ofthe invention by standard techniques known in the art, such assite-directed mutagenesis and PCR-mediated mutagenesis. Conservativeamino acid substitutions are ones in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody accordingto at least some embodiments of the invention can be replaced with otheramino acid residues from the same side chain family and the alteredantibody can be tested for retained function (i.e., the functions setforth in (c) through (j) above) using the functional assays describedherein.

Antibodies that Bind to the Same Epitope as Anti-LY6G6F, Anti-VSIG10,Anti-TMEM25 or Anti-LSR According to at Least Some Embodiments of theInvention.

In another embodiment, the invention provides antibodies that bind topreferred epitopes on human LY6G6F, VSIG10, TMEM25 or LSR which possessdesired functional properties such as modulation of B7 co-stimulationand related functions. Other antibodies with desired epitope specificitymay be selected and will have the ability to cross-compete for bindingto LY6G6F, VSIG10, TMEM25 or LSR antigen with the desired antibodies.

Engineered and Modified Antibodies

An antibody according to at least some embodiments of the inventionfurther can be prepared using an antibody having one or more of the VHand/or VL sequences derived from an anti-LY6G6F, anti-VSIG10,anti-TMEM25 or anti-LSR antibody starting material to engineer amodified antibody, which modified antibody may have altered propertiesfrom the starting antibody. An antibody can be engineered by modifyingone or more residues within one or both variable regions (i.e., VHand/or VL), for example within one or more CDR regions and/or within oneor more framework regions. Additionally or alternatively, an antibodycan be engineered by modifying residues within the constant regions, forexample to alter the effector functions of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Suitable framework sequences can be obtained from public DNA databasesor published references that include germline antibody gene sequences.For example, germline DNA sequences for human heavy and light chainvariable region genes can be found in the “VBase” human germlinesequence database (available on the Internet), as well as in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242; Tomlinson, I. M., et al. (1992) “The Repertoire of HumanGermline VH Sequences Reveals about Fifty Groups of VH Segments withDifferent Hypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, J. P.L. et al. (1994) “A Directory of Human Germ-line VH Segments Reveals aStrong Bias in their Usage” Eur. J Immunol. 24:827-836; the contents ofeach of which are expressly incorporated herein by reference.

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR 1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutations and the effecton antibody binding, or other functional property of interest, can beevaluated in appropriate in vitro or in vivo assays. Preferablyconservative modifications (as discussed above) are introduced. Themutations may be amino acid substitutions, additions or deletions, butare preferably substitutions. Moreover, typically no more than one, two,three, four or five residues within a CDR region are altered.

Engineered antibodies according to at least some embodiments of theinvention include those in which modifications have been made toframework residues within VH and/or VL, e.g. to improve the propertiesof the antibody. Typically such framework modifications are made todecrease the immunogenicity of the antibody. For example, one approachis to “backmutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody that has undergonesomatic mutation may contain framework residues that differ from thegermline sequence from which the antibody is derived. Such residues canbe identified by comparing the antibody framework sequences to thegermline sequences from which the antibody is derived.

In addition or alternative to modifications made within the framework orCDR regions, antibodies according to at least some embodiments of theinvention may be engineered to include modifications within the Fcregion, typically to alter one or more functional properties of theantibody, such as serum half-life, complement fixation, Fc receptorbinding, and/or antigen-dependent cellular cytotoxicity. Furthermore, anantibody according to at least some embodiments of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Suchembodiments are described further below. The numbering of residues inthe Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322 can be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered Clq binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcy receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for Fc gamma RI, Fc gamma RII,Fc gammaRIII and FcRn have been mapped and variants with improvedbinding have been described (see Shields, R. L. et al. (2001) J. Biol.Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298,333, 334 and 339 are shown to improve binding to FcyRIII. Additionally,the following combination mutants are shown to improve Fcgamma.RIIIbinding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improvebinding to FcRn and increase antibody circulation half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies according to at least some embodiments of theinvention to thereby produce an antibody with altered glycosylation. Forexample, the cell lines Ms704, Ms705, and Ms709 lack thefucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), suchthat antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8.−/− celllines are created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al.(2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 byHanai et al. describes a cell line with a functionally disrupted FUT8gene, which encodes a fucosyl transferase, such that antibodiesexpressed in such a cell line exhibit hypofucosylation by reducing oreliminating the alpha 1,6 bond-related enzyme. Hanai et al. alsodescribe cell lines which have a low enzyme activity for adding fucoseto the N-acetylglucosamine that binds to the Fc region of the antibodyor does not have the enzyme activity, for example the rat myeloma cellline YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Prestadescribes a variant CHO cell line, Lec13 cells, with reduced ability toattach fucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody may be cleaved offusing a fucosidase enzyme. For example, the fucosidasealpha-L-fucosidase removes fucosyl residues from antibodies (Tarentino,A. L. et al. (1975) Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies according to at least some embodiments of theinvention. See for example, EP 0 154 316 by Nishimura et al. and EP 0401 384 by Ishikawa et al.

Methods of Engineering Antibodies

As discussed above, anti-LY6G6F, anti-VSIG10, anti-TMEM25 or anti-LSRantibodies having VH and VK sequences disclosed herein can be used tocreate new anti-LY6G6F, anti-VSIG10, anti-TMEM25 or anti-LSR antibodies,respectively, by modifying the VH and/or VL sequences, or the constantregions attached thereto. Thus, in another aspect according to at leastsome embodiments of the invention, the structural features of ananti-LY6G6F, anti-VSIG10, anti-TMEM25 or anti-LSR antibody according toat least some embodiments of the invention, are used to createstructurally related anti-LY6G6F, anti-VSIG10, anti-TMEM25 or anti-LSRantibodies that retain at least one functional property of theantibodies according to at least some embodiments of the invention, suchas binding to human LY6G6F, VSIG10, TMEM25 or LSR, respectively. Forexample, one or more CDR regions of one LY6G6F, VSIG10, TMEM25 or LSRantibody or mutations thereof, can be combined recombinantly with knownframework regions and/or other CDRs to create additional,recombinantly-engineered, anti-LY6G6F, anti-VSIG10, anti-TMEM25 oranti-LSR antibodies according to at least some embodiments of theinvention, as discussed above. Other types of modifications includethose described in the previous section. The starting material for theengineering method is one or more of the VH and/or VK sequences providedherein, or one or more CDR regions thereof. To create the engineeredantibody, it is not necessary to actually prepare (i.e., express as aprotein) an antibody having one or more of the VH and/or VK sequencesprovided herein, or one or more CDR regions thereof. Rather, theinformation contained in the sequences is used as the starting materialto create a “second generation” sequences derived from the originalsequences and then the “second generation” sequences is prepared andexpressed as a protein.

Standard molecular biology techniques can be used to prepare and expressaltered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequences isone that retains one, some or all of the functional properties of theanti-LY6G6F, anti-VSIG10, anti-TMEM25 or anti-LSR antibodies,respectively, produced by methods and with sequences provided herein,which functional properties include binding to LY6G6F, VSIG10, TMEM25 orLSR antigen with a specific KD level or less and/or modulating B7costimulation and/or selectively binding to desired target cells such asfor example melanoma, cancers of liver, renal, brain, breast, colon,lung, ovary, pancreas, prostate, stomach, multiple myeloma andhematopoietic cancer, including but not limited to lymphoma (Hodgkin'sand non Hodgkin's), acute and chronic lymphoblastic leukemia and acuteand chronic myeloid leukemia, that express LY6G6F, VSIG10, TMEM25 and/orLSR antigen.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein.

In certain embodiments of the methods of engineering antibodiesaccording to at least some embodiments of the invention, mutations canbe introduced randomly or selectively along all or part of ananti-LY6G6F, anti-VSIG10, anti-TMEM25 or anti-LSR antibody codingsequence and the resulting modified anti-LY6G6F, anti-VSIG10,anti-TMEM25 or anti-LSR antibodies can be screened for binding activityand/or other desired functional properties.

Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

Nucleic Acid Molecules Encoding Antibodies

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies according to at least some embodiments of theinvention. The nucleic acids may be present in whole cells, in a celllysate, or in a partially purified or substantially pure form. A nucleicacid is “isolated” or “rendered substantially pure” when purified awayfrom other cellular components or other contaminants, e.g., othercellular nucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. (1987) Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid according toat least some embodiments of the invention can be, for example, DNA orRNA and may or may not contain intronic sequences. In a preferredembodiment, the nucleic acid is a cDNA molecule.

Nucleic acids according to at least some embodiments of the inventioncan be obtained using standard molecular biology techniques. Forantibodies expressed by hybridomas (e.g., hybridomas prepared fromtransgenic mice carrying human immunoglobulin genes as described furtherbelow), cDNAs encoding the light and heavy chains of the antibody madeby the hybridoma can be obtained by standard PCR amplification or cDNAcloning techniques. For antibodies obtained from an immunoglobulin genelibrary (e.g., using phage display techniques), nucleic acid encodingthe antibody can be recovered from the library.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker.

The term “operatively linked”, as used in this context, is intended tomean that the two DNA fragments are joined such that the amino acidsequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., el al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Production of Anti-LY6G6F, Anti-VSIG10, Anti-TMEM25 or Anti-LSRMonoclonal Antibodies

Monoclonal antibodies (mAbs) of the present invention can be produced bya variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256:495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

A preferred animal system for preparing hybridomas is the murine system.

Hybridoma production in the mouse is a very well-established procedureImmunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g. human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.).

According to at least some embodiments of the invention, the antibodiesare human monoclonal antibodies. Such human monoclonal antibodiesdirected against LY6G6F, VSIG10, TMEM25 and/or LSR can be generatedusing transgenic or transchromosomic mice carrying parts of the humanimmune system rather than the mouse system. These transgenic andtranschromosomic mice include mice referred to herein as the HuMAbMouse® and KM Mouse®, respectively, and are collectively referred toherein as “human Ig mice.” The HuMAb Mouse™. (Medarex. Inc.) containshuman immunoglobulin gene miniloci that encode unrearranged human heavy(.mu. and .gamma.) and .kappa. light chain immunoglobulin sequences,together with targeted mutations that inactivate the endogenous .mu. and.kappa. chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474):856-859). Accordingly, the mice exhibit reduced expression of mouse IgMor .kappa., and in response to immunization, the introduced human heavyand light chain transgenes undergo class switching and somatic mutationto generate high affinity human IgGkappa. monoclonal (Lonberg, N. et al.(1994), supra; reviewed in Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.Acad. Sci. 764:536-546). The preparation and use of the HuMab Mouse®,and the genomic modifications carried by such mice, is further describedin Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen,J. et al. (1993) International Immunology 5:647-656; Tuaillon et al.(1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993)Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830;Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al.(1994) International Immunology 6:579-591; and Fishwild, D. et al.(1996) Nature Biotechnology 14: 845-851, the contents of all of whichare hereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies according to at least someembodiments of the invention can be raised using a mouse that carrieshuman immunoglobulin sequences on transgenes and transchomosomes, suchas a mouse that carries a human heavy chain transgene and a human lightchain transchromosome. Such mice, referred to herein as “KM Mice™.”, aredescribed in detail in PCT Publication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSR antibodiesaccording to at least some embodiments of the invention. For example, analternative transgenic system referred to as the Xenomouse (Abgenix,Inc.) can be used; such mice are described in, for example, U.S. Pat.Nos. 5,939,598; 6,075,181; 6,114,598; 6, 150,584 and 6,162,963 toKucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSR antibodiesaccording to at least some embodiments of the invention. For example,mice carrying both a human heavy chain transchromosome and a human lightchain transchromosome, referred to as “TC mice” can be used; such miceare described in Tomizuka et al. (2000) Proc. Natl. Acad Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-LY6G6F,anti-VSIG10, anti-TMEM25 and/or anti-LSR antibodies according to atleast some embodiments of the invention.

Human monoclonal antibodies according to at least some embodiments ofthe invention can also be prepared using phage display methods forscreening libraries of human immunoglobulin genes. Such phage displaymethods for isolating human antibodies are established in the art. Seefor example: U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No.5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 toDower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies according to at least some embodiments ofthe invention can also be prepared using SCID mice into which humanimmune cells have been reconstituted such that a human antibody responsecan be generated upon immunization. Such mice are described in, forexample, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies according to atleast some embodiments of the invention, such mice can be immunized witha purified or enriched preparation of LY6G6F, VSIG10, TMEM25 and/or LSRantigen and/or recombinant LY6G6F, VSIG10, TMEM25 and/or LSR, or LY6G6F,VSIG10, TMEM25 and/or LSR fusion protein, as described by Lonberg, N. etal. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851; and PCT Publication WO 98/24884 and WO01/14424. Preferably, the mice will be 6-16 weeks of age upon the firstinfusion. For example, a purified or recombinant preparation (5-50.mu.g) of LY6G6F, VSIG10, TMEM25 and/or LSR antigen can be used toimmunize the human Ig mice intraperitoneally.

Prior experience with various antigens by others has shown that thetransgenic mice respond when initially immunized intraperitoneally (IP)with antigen in complete Freund's adjuvant, followed by every other weekIP immunizations (up to a total of 6) with antigen in incompleteFreund's adjuvant. However, adjuvants other than Freund's are also foundto be effective. In addition, whole cells in the absence of adjuvant arefound to be highly immunogenic. The immune response can be monitoredover the course of the immunization protocol with plasma samples beingobtained by retroorbital bleeds. The plasma can be screened by ELISA (asdescribed below), and mice with sufficient titers of anti-LY6G6F,anti-VSIG10, anti-TMEM25 and/or anti-LSR human immunoglobulin can beused for fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. Usually both HCo7 andHCo12 strains are used. In addition, both HCo7 and HCo 12 transgene canbe bred together into a single mouse having two different human heavychain transgenes (HCo7/HCo 12). Alternatively or additionally, the KMMouse® strain can be used.

Generation of Hybridomas Producing Human Monoclonal Antibodies

To generate hybridomas producing human monoclonal antibodies accordingto at least some embodiments of the invention, splenocytes and/or lymphnode cells from immunized mice can be isolated and fused to anappropriate immortalized cell line, such as a mouse myeloma cell line.The resulting hybridomas can be screened for the production ofantigen-specific antibodies. For example, single cell suspensions ofsplenic lymphocytes from immunized mice can be fused to one-sixth thenumber of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL1580) with 50% PEG. Cells are plated at approximately 2×10-5 in flatbottom microtiter plate, followed by a two week incubation in selectivemedium containing 20% fetal Clone Serum, 18% “653” conditioned media, 5%origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after thefusion). After approximately two weeks, cells can be cultured in mediumin which the HAT is replaced with HT. Individual wells can then bescreened by ELISA for human monoclonal IgM and IgG antibodies. Onceextensive hybridoma growth occurs, medium can be observed usually after10-14 days. The antibody secreting hybridomas can be replated, screenedagain, and if still positive for human IgG, the monoclonal antibodiescan be subcloned at least twice by limiting dilution. The stablesubclones can then be cultured in vitro to generate small amounts ofantibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80 degrees C.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies according to at least some embodiments according to at leastsome embodiments of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the VH segmentis operatively linked to the CH segments within the vector and the VKsegment is operatively linked to the CL segment within the vector.Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors according to at least some embodiments of the invention carryregulatory sequences that control the expression of the antibody chaingenes in a host cell. The term “regulatory sequence” is intended toinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals) that control the transcription ortranslation of the antibody chain genes. Such regulatory sequences aredescribed, for example, in Goeddel (Gene Expression Technology. Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences, maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV), SimianVirus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may beused, such as the ubiquitin promoter or .beta.-globin promoter. Stillfurther, regulatory elements composed of sequences from differentsources, such as the SR alpha. promoter system, which contains sequencesfrom the SV40 early promoter and the long terminal repeat of human Tcell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol.8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors according to at least some embodiments ofthe invention may carry additional sequences, such as sequences thatregulate replication of the vector in host cells (e.g., origins ofreplication) and selectable marker genes. The selectable marker genefacilitates selection of host cells into which the vector has beenintroduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Preferred selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vectorsencoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies according to at least someembodiments of the invention in either prokaryotic or eukaryotic hostcells, expression of antibodies in eukaryotic cells, and most preferablymammalian host cells, is the most preferred because such eukaryoticcells, and in particular mammalian cells, are more likely thanprokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesaccording to at least some embodiments of the invention include ChineseHamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlauband ChasM, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with aDHFR selectable marker, e.g., as described in R. J. Kaufman and P. A.Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells andSP2 cells. In particular, for use with NSO myeloma cells, anotherpreferred expression system is the GS gene expression system disclosedin WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Characterization of Antibody Binding to Antigen

Antibodies according to at least some embodiments of the invention canbe tested for binding to LY6G6F, VSIG10, TMEM25 and/or LSR by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified LY6G6F, VSIG10, TMEM25 and/or LSR at 0.25 .mu.g/ml in PBS, andthen blocked with 5% bovine serum albumin in PBS. Dilutions of antibody(e.g., dilutions of plasma from —immunized mice) are added to each welland incubated for 1-2 hours at 37 degrees C. The plates are washed withPBS/Tween and then incubated with secondary reagent (e.g., for humanantibodies, a goat-anti-human IgG Fc-specific polyclonal reagent)conjugated to alkaline phosphatase for 1 hour at 37 degrees C. Afterwashing, the plates are developed with pNPP substrate (1 mg/ml), andanalyzed at OD of 405-650. Preferably, mice which develop the highesttiters will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with LY6G6F, VSIG10, TMEM25and/or LSR immunogen. Hybridomas that bind with high avidity to LY6G6F,VSIG10, TMEM25 and/or LSR are subcloned and further characterized. Oneclone from each hybridoma, which retains the reactivity of the parentcells (by ELISA), can be chosen for making a 5-10 vial cell bank storedat −140 degrees C., and for antibody purification.

To purify anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSRantibodies, selected hybridomas can be grown in two-liter spinner-flasksfor monoclonal antibody purification. Supernatants can be filtered andconcentrated before affinity chromatography with protein A-sepharose(Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gelelectrophoresis and high performance liquid chromatography to ensurepurity. The buffer solution can be exchanged into PBS, and theconcentration can be determined by OD280 using 1.43 extinctioncoefficient. The monoclonal antibodies can be aliquoted and stored at−80 degrees C.

To determine if the selected anti-LY6G6F, anti-VSIG10, anti-TMEM25and/or anti-LSRmonoclonal antibodies bind to unique epitopes, eachantibody can be biotinylated using commercially available reagents(Pierce, Rockford, Ill.). Competition studies using unlabeled monoclonalantibodies and biotinylated monoclonal antibodies can be performed usingLY6G6F, VSIG10, TMEM25 and/or LSR coated-ELISA plates as describedabove. Biotinylated mAb binding can be detected with astrep-avidin-alkaline phosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 .mu.g/ml ofanti-human immunoglobulin overnight at 4 degrees C. After blocking with1% BSA, the plates are reacted with 1 mug/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

Anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSRhuman IgGs can befurther tested for reactivity with LY6G6F, VSIG10, TMEM25 and/or LSRantigen, respectively, by Western blotting. Briefly, LY6G6F, VSIG10,TMEM25 and/or LSRantigen can be prepared and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis. After electrophoresis, theseparated antigens are transferred to nitrocellulose membranes, blockedwith 10% fetal calf serum, and probed with the monoclonal antibodies tobe tested. Human IgG binding can be detected using anti-human IgGalkaline phosphatase and developed with BCIP/NBT substrate tablets(Sigma Chem. Co., St. Louis, Mo.).

Alternative Scaffolds

According to at least some embodiments the invention relates to proteinscaffolds with specificities and affinities in a range similar tospecific antibodies. According to at least some embodiments the presentinvention relates to an antigen-binding construct comprising a proteinscaffold which is linked to one or more epitope-binding domains. Suchengineered protein scaffolds are usually obtained by designing a randomlibrary with mutagenesis focused at a loop region or at an otherwisepermissible surface area and by selection of variants against a giventarget via phage display or related techniques. According to at leastsome embodiments the invention relates to alternative scaffoldsincluding, but not limited to, anticalins, DARPins, Armadillo repeatproteins, protein A, lipocalins, fibronectin domain, ankyrin consensusrepeat domain, thioredoxin, chemically constrained peptides and thelike. According to at least some embodiments the invention relates toalternative scaffolds that are used as therapeutic agents for treatmentof cancer, autoimmune and infectious diseases as well as for in vivodiagnostics.

According to at least some embodiments the invention further provides apharmaceutical composition comprising an antigen binding construct asdescribed herein a pharmaceutically acceptable carrier.

The term ‘Protein Scaffold’ as used herein includes but is not limitedto an immunoglobulin (Ig) scaffold, for example an IgG scaffold, whichmay be a four chain or two chain antibody, or which may comprise onlythe Fc region of an antibody, or which may comprise one or more constantregions from an antibody, which constant regions may be of human orprimate origin, or which may be an artificial chimera of human andprimate constant regions. Such protein scaffolds may compriseantigen-binding sites in addition to the one or more constant regions,for example where the protein scaffold comprises a full IgG. Suchprotein scaffolds will be capable of being linked to other proteindomains, for example protein domains which have antigen-binding sites,for example epitope-binding domains or ScFv domains.

A “domain” is a folded protein structure which has tertiary structureindependent of the rest of the protein. Generally, domains areresponsible for discrete functional properties of proteins and in manycases may be added, removed or transferred to other proteins withoutloss of function of the remainder of the protein and/or of the domain. A“single antibody variable domain” is a folded polypeptide domaincomprising sequences characteristic of antibody variable domains. Ittherefore includes complete antibody variable domains and modifiedvariable domains, for example, in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least the binding activity andspecificity of the full-length domain.

The phrase “immunoglobulin single variable domain” refers to an antibodyvariable domain (VH, V HH, V L) that specifically binds an antigen orepitope independently of a different V region or domain. Animmunoglobulin single variable domain can be present in a format (e.g.,homo- or hetero-multimer) with other, different variable regions orvariable domains where the other regions or domains are not required forantigen binding by the single immunoglobulin variable domain (i.e.,where the immunoglobulin single variable domain binds antigenindependently of the additional variable domains). A “domain antibody”or “dAb” is the same as an “immunoglobulin single variable domain” whichis capable of binding to an antigen as the term is used herein. Animmunoglobulin single variable domain may be a human antibody variabledomain, but also includes single antibody variable domains from otherspecies such as rodent (for example, as disclosed in WO 00/29004), nurseshark and Camelid V HH dAbs. Camelid V HH are immunoglobulin singlevariable domain polypeptides that are derived from species includingcamel, llama, alpaca, dromedary, and guanaco, which produce heavy chainantibodies naturally devoid of light chains. Such V HH domains may behumanised according to standard techniques available in the art, andsuch domains are still considered to be “domain antibodies” according tothe invention. As used herein “VH includes camelid V HH domains. NARYare another type of immunoglobulin single variable domain which wereidentified in cartilaginous fish including the nurse shark. Thesedomains are also known as Novel Antigen Receptor variable region(commonly abbreviated to V(NAR) or NARY). For further details see MoI.Immunol. 44, 656-665 (2006) and US20050043519A.

The term “epitope-binding domain” refers to a domain that specificallybinds an antigen or epitope independently of a different V region ordomain, this may be a domain antibody (dAb), for example a human,camelid or shark immunoglobulin single variable domain or itmay be adomain which is a derivative of a scaffold selected from the groupconsisting of CTLA-4 (Evibody); lipocalin; Protein A derived moleculessuch as Z-domain of Protein A (Affibody, SpA), A-domain(Avimer/Maxibody); Heat shock proteins such as GroEI and GroES;transferrin (trans-body); ankyrin repeat protein (DARPin); peptideaptamer; C-type lectin domain (Tetranectin); human &#947;—crystallin andhuman ubiquitin (affilins); PDZ domains; scorpion toxinkunitz typedomains of human protease inhibitors; Armadillo repeat proteins,thioredoxin, and fibronectin (adnectin); which has been subjected toprotein engineering in order to obtain binding to a ligand other thanthe natural ligand.

Loops corresponding to CDRs of antibodies can be substituted withheterologous sequence to confer different binding properties i.e.Evibodies. For further details see Journal of Immunological Methods 248(1-2), 31-45 (2001) Lipocalins are a family of extracellular proteinswhich transport small hydrophobic molecules such as steroids, bilins,retinoids and lipids. They have a rigid secondary structure with a numerof loops at the open end of the conical structure which can beengineered to bind to different target antigens. Anticalins are between160-180 amino acids in size, and are derived from lipocalins. Forfurther details see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Pat.No. 7,250,297B1 and US20070224633. An affibody is a scaffold derivedfrom Protein A of Staphylococcus aureus which can be engineered to bindto antigen. The domain consists of a three-helical bundle ofapproximately 58 amino acids. Libraries have been generated byrandomisation of surface residues. For further details see Protein Eng.Des. SeI. 17, 455-462 (2004) and EP1641818A1 Avimers are multidomainproteins derived from the A-domain scaffold family. The native domainsof approximately 35 amino acids adopt a defined disulphide bondedstructure. Diversity is generated by shuffling of the natural variationexhibited by the family of A-domains. For further details see NatureBiotechnology 23(12), 1556-1561 (2005) and Expert Opinion onInvestigational Drugs 16(6), 909-917 (June 2007) A transferrin is amonomeric serum transport glycoprotein. Transferrins can be engineeredto bind different target antigens by insertion of peptide sequences in apermissive surface loop. Examples of engineered transferrin scaffoldsinclude the Trans-body. For further details see J. Biol. Chem 274,24066-24073 (1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrinwhich is a family of proteins that mediate attachment of integralmembrane proteins to the cytoskeleton. A single ankyrin repeat is a 33residue motif consisting of two alpha helices;—beta turn. They can beengineered to bind different target antigens by randomising residues inthe first alpha-helix and a beta-turn of each repeat. Their bindinginterface can be increased by increasing the number of modules (a methodof affinity maturation). For further details see J. MoI. Biol. 332,489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. MoI. Biol. 369,1015-1028 (2007) and US20040132028A1.

Fibronectin is a scaffold which can be engineered to bind to antigen.Adnectins consists of a backbone of the natural amino acid sequence ofthe 10th domain of the 15 repeating units of human fibronectin type III(FN3). Three loops at one end of the beta;—sandwich can be engineered toenable an Adnectin to specifically recognize a therapeutic target ofinterest. For further details see Protein Eng. Des. SeI. 18, 435-444(2005), US200801 39791, WO2005056764 and U.S. Pat. No. 6,818,418B1.

Peptide aptamers are combinatorial recognition molecules that consist ofa constant scaffold protein, typically thioredoxin (TrxA) which containsa constrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5. 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBl and conotoxin and knottins. Themicroproteins have a loop which can be engineered to include upto 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

Other epitope binding domains include proteins which have been used as ascaffold to engineer different target antigen binding properties includehuman &#947;beta-crystallin and human ubiquitin (affilins), kunitz typedomains of human protease inhibitors, PDZ-domains of the Ras-bindingprotein AF-6, scorpion toxins (charybdotoxin), C-type lectin domain(tetranectins) are reviewed in Chapter 7—Non-Antibody Scaffolds fromHandbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) andProtein Science 15:14-27 (2006). Epitope binding domains of the presentinvention could be derived from any of these alternative proteindomains.

Conjugates or Immunoconjugates

The present invention encompasses conjugates for use in immune therapycomprising the LY6G6F, VSIG10, TMEM25 and/or LSR antigen and solubleportions thereof including the ectodomain or portions or variantsthereof. For example the invention encompasses conjugates wherein theECD of the LY6G6F, VSIG10, TMEM25 and/or LSR antigen is attached to animmunoglobulin or fragment thereof. The invention contemplates the usethereof for promoting or inhibiting LY6G6F, VSIG10, TMEM25 and/or LSRantigen activities such as immune costimulation and the use thereof intreating transplant, autoimmune, and cancer indications describedherein.

In another aspect, the present invention features antibody-drugconjugates (ADCs), used for example for treatment of cancer, consistingof an antibody (or antibody fragment such as a single-chain variablefragment [scFv]) linked to a payload drug (often cytotoxic). Theantibody causes the ADC to bind to the target cancer cells. Often theADC is then internalized by the cell and the drug is released into thecell. Because of the targeting, the side effects are lower and give awider therapeutic window. Hydrophilic linkers (e.g., PEG4Mal) helpprevent the drug being pumped out of resistant cancer cells through MDR(multiple drug resistance) transporters. ADCs based on cleavable linkersare thought to have a less favorable therapeutic window, but targets(tumor cell surface antigens) that do not get internalized efficientlyseem more suitable for cleavable linkers.

In another aspect, the present invention features immunoconjugatescomprising an anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSRantibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g., an immunosuppressant) or aradiotoxin. Such conjugates are referred to herein as “immunoconjugates”Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody according to at least some embodiments of theinvention include duocarmycins, calicheamicins, maytansines andauristatins, and derivatives thereof. An example of a calicheamicinantibody conjugate is commercially available (Mylotarg™; Wyeth).

Cytotoxins can be conjugated to antibodies according to at least someembodiments of the invention using linker technology available in theart. Examples of linker types that have been used to conjugate acytotoxin to an antibody include, but are not limited to, hydrazones,thioethers, esters, disulfides and peptide-containing linkers. A linkercan be chosen that is, for example, susceptible to cleavage by low pHwithin the lysosomal compartment or susceptible to cleavage byproteases, such as proteases preferentially expressed in tumor tissuesuch as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine 131, indium 111,yttrium 90 and lutetium 177. Methods for preparing radioimmunconjugatesare established in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin (IDEC Pharmaceuticals) andBexxar. (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies according to at leastsome embodiments of the invention.

The antibody conjugates according to at least some embodiments of theinvention can be used to modify a given biological response, and thedrug moiety is not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, an enzymatically active toxin, or active fragmentthereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin; a protein such as tumor necrosis factor or interferon-.gamma.;or, biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSRantibody, or a fragment thereof, according to at least some embodimentsof the invention. An antibody according to at least some embodiments ofthe invention, or antigen-binding portions thereof, can be derivatizedor linked to another functional molecule, e.g., another peptide orprotein (e.g., another antibody or ligand for a receptor) to generate abispecific molecule that binds to at least two different binding sitesor target molecules. The antibody according to at least some embodimentsof the invention may in fact be derivatized or linked to more than oneother functional molecule to generate multispecific molecules that bindto more than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific moleculeaccording to at least some embodiments of the invention, an antibody canbe functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for LY6G6F, VSIG10,TMEM25 and/or LSR and a second binding specificity for a second targetepitope. According to at least some embodiments of the invention, thesecond target epitope is an Fc receptor, e.g., human Fc gamma RI (CD64)or a human Fc alpha receptor (CD89). Therefore, the invention includesbispecific molecules capable of binding both to Fc gamma. R, Fc alpha Ror Fc epsilon R expressing effector cells (e.g., monocytes, macrophagesor polymorphonuclear cells (PMNs)), and to target cells expressingLY6G6F, VSIG10, TMEM25 and/or LSR, respectively. These bispecificmolecules target LY6G6F, VSIG10, TMEM25 and/or LSR expressing cells toeffector cell and trigger Fc receptor-mediated effector cell activities,such as phagocytosis of an LY6G6F, VSIG10, TMEM25 and/or LSR expressingcells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokinerelease, or generation of superoxide anion.

According to at least some embodiments of the invention in which thebispecific molecule is multispecific, the molecule can further include athird binding specificity, in addition to an anti-Fc binding specificityand an anti-6f binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell.

The “anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the Fc receptor or target cellantigen. The “anti-enhancement factor portion” can bind an Fc receptoror a target cell antigen. Alternatively, the anti-enhancement factorportion can bind to an entity that is different from the entity to whichthe first and second binding specificities bind. For example, theanti-enhancement factor portion can bind a cytotoxic T-cell (e.g., viaCD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that resultsin an increased immune response against the target cell).

According to at least some embodiments of the invention, the bispecificmolecules comprise as a binding specificity at least one antibody, or anantibody fragment thereof, including, e.g., an Fab, Fab′, F(ab′).sub.2,Fv, or a single chain Fv. The antibody may also be a light chain orheavy chain dimer, or any minimal fragment thereof such as a Fv or asingle chain construct as described in Ladner et al. U.S. Pat. No.4,946,778, the contents of which is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcy receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight .gamma.-chain genes located on chromosome 1.These genes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fc.gamma. receptor classes: Fcgamma R1 (CD64), Fc gamma RII(CD32), and Fc gamma.RIII (CD 16). In onepreferred embodiment, the Fc gamma. receptor a human high affinityFc.gamma RI. The human Fc gammaRI is a 72 kDa molecule, which shows highaffinity for monomeric IgG (10 8-10-9 M.-1).

The production and characterization of certain preferred anti-Fc gamma.monoclonal antibodies are described by Fanger et al. in PCT PublicationWO 88/00052 and in U.S. Pat. No. 4,954,617, the teachings of which arefully incorporated by reference herein. These antibodies bind to anepitope of Fc.gamma.R1, FcyRII or FcyRIII at a site which is distinctfrom the Fc.gamma. binding site of the receptor and, thus, their bindingis not blocked substantially by physiological levels of IgG. Specificanti-Fc.gamma.RI antibodies useful in this invention are mAb 22, mAb 32,mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32 is availablefrom the American Type Culture Collection, ATCC Accession No. HB9469. Inother embodiments, the anti-Fcy receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol.155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibodyproducing cell line is deposited at the American Type Culture Collectionunder the designation HAO22CLI and has the accession no. CRL 11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (Fc alpha.RI(CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one alpha.-gene (Fcalpha.RI) located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 10 kDa.

Fc.alpha.RI (CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. Fc alpha RI has medium affinity (Approximately 5×10-7 M-1)for both IgA1 and IgA2, which is increased upon exposure to cytokinessuch as G-CSF or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews inImmunology 16:423-440). Four FcaRI-specific monoclonal antibodies,identified as A3, A59, A62 and A77, which bind Fc.alpha.RI outside theIgA ligand binding domain, have been described (Monteiro, R. C. et al.(1992) J. Immunol. 148:1764).

Fc. alpha. RI and Fc gamma. RI are preferred trigger receptors for usein the bispecific molecules according to at least some embodiments ofthe invention because they are (1) expressed primarily on immuneeffector cells, e.g., monocytes, PMNs, macrophages and dendritic cells;(2) expressed at high levels (e.g., 5,000-100,000 per cell); (3)mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4)mediate enhanced antigen presentation of antigens, includingself-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules according to at least someembodiments of the invention are murine, chimeric and humanizedmonoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSR bindingspecificities, using methods known in the art. For example, each bindingspecificity of the bispecific molecule can be generated separately andthen conjugated to one another. When the binding specificities areproteins or peptides, a variety of coupling or cross-linking agents canbe used for covalent conjugation. Examples of cross-linking agentsinclude protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate(SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB),o-phenylenedimaleimide (oPDM),N-succinimidyl-3-(2-pyridyld-ithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAbXmAb, mAbXFab,FabXF(ab′)2 or ligandXFab fusion protein. A bispecific moleculeaccording to at least some embodiments of the invention can be a singlechain molecule comprising one single chain antibody and a bindingdeterminant, or a single chain bispecific molecule comprising twobinding determinants. Bispecific molecules may comprise at least twosingle chain molecules. Methods for preparing bispecific molecules aredescribed for example in U.S. Pat. No. 5,260,203; U.S. Pat. No.5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat.No. 5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S.Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a gamma. counter or ascintillation counter or by autoradiography.

Protein Modifications

Fusion Proteins

According to at least some embodiments, LY6G6F, VSIG10, TMEM25 and/orLSR fusion polypeptides have a first fusion partner comprising all or apart of a LY6G6F, VSIG10, TMEM25 and/or LSR protein fused to a secondpolypeptide directly or via a linker peptide sequence or a chemicallinker useful to connect the two proteins. The LY6G6F, VSIG10, TMEM25and/or LSR polypeptide may optionally be fused to a second polypeptideto form a fusion protein as described herein. The presence of the secondpolypeptide can alter the solubility, stability, affinity and/or valencyof the LY6G6F, VSIG10, TMEM25 and/or LSR fusion polypeptide. As usedherein, “valency” refers to the number of binding sites available permolecule. In one embodiment the second polypeptide is a polypeptide froma different source or different protein.

According to at least some embodiments, the LY6G6F, VSIG10, TMEM25and/or LSR protein or fragment is selected for its activity for thetreatment of immune related disorder and/or infectious disorder, and/orcancer as described herein.

In one embodiment, the second polypeptide contains one or more domainsof an immunoglobulin heavy chain constant region, preferably having anamino acid sequence corresponding to the hinge, CH2 and CH3 regions of ahuman immunoglobulin Cγ1, Cγ2, Cγ3 or Cγ4 chain or to the hinge, CH2 andCH3 regions of a murine immunoglobulin Cγ2a chain. SEQ ID NO: 70provides exemplary sequence for the hinge, CH2 and CH3 regions of ahuman immunoglobulin Cγ1.

According to at least some embodiments, the fusion protein is a dimericfusion protein. In an optional dimeric fusion protein, the dimer resultsfrom the covalent bonding of Cys residue in the hinge region of two ofthe Ig heavy chains that are the same Cys residues that are disulfidelinked in dimerized normal Ig heavy chains. Such proteins are referredto as LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, fragments orfusion proteins thereof.

In one embodiment, the immunoglobulin constant domain may contain one ormore amino acid insertions, deletions or substitutions that enhancebinding to specific cell types, increase the bioavailablity, or increasethe stability of the LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides,fusion proteins, or fragments thereof. Suitable amino acid substitutionsinclude conservative and non-conservative substitutions, as describedabove.

The fusion proteins optionally contain a domain that functions todimerize or multimerize two or more fusion proteins. Thepeptide/polypeptide linker domain can either be a separate domain, oralternatively can be contained within one of the other domains (LY6G6F,VSIG10, TMEM25 and/or LSR polypeptide or second polypeptide) of thefusion protein. Similarly, the domain that functions to dimerize ormultimerize the fusion proteins can either be a separate domain, oralternatively can be contained within one of the other domains (LY6G6F,VSIG10, TMEM25 and/or LSR polypeptide, second polypeptide orpeptide/polypeptide linker domain) of the fusion protein. In oneembodiment, the dimerization/multimerization domain and thepeptide/polypeptide linker domain are the same. Further specific,illustrative and non-limiting examples of dimerization/multimerizationdomains and linkers are given below.

Fusion proteins disclosed herein according to at least some embodimentsof the present invention are of formula I: N-R1-R2-R3-C wherein “N”represents the N-terminus of the fusion protein, “C” represents theC-terminus of the fusion protein. In the further embodiment, “R1” is aLY6G6F, VSIG10, TMEM25 and/or LSR polypeptide, “R2” is an optionalpeptide/polypeptide or chemical linker domain, and “R3” is a secondpolypeptide. Alternatively, R3 may be a LY6G6F, VSIG10, TMEM25 and/orLSR polypeptide and R1 may be a second polypeptide. Various non-limitingexamples of linkers are described in greater detail below.

Optionally, the fusion protein comprises the LY6G6F, VSIG10, TMEM25and/or LSR polypeptide fragments as described herein, fused, optionallyby a linker peptide of one or more amino acids (e.g. GS) to one or more“half-life extending moieties”. A “half-life extending moiety” is anymoiety, for example, a polypeptide, small molecule or polymer, that,when appended to protein, extends the in vivo half-life of that proteinin the body of a subject (e.g., in the plasma of the subject). Forexample, a half-life extending moiety is, in an embodiment of theinvention, polyethylene glycol (PEG), monomethoxy PEG (mPEG) or animmunoglobulin (Ig). In an embodiment of the invention, PEG is a 5, 10,12, 20, 30, 40 or 50 kDa moiety or larger or comprises about 12000ethylene glycol units (PEG12000).

The fusion protein may also optionally be prepared by chemical syntheticmethods and the “join” effected chemically, either during synthesis orpost-synthesis. Cross-linking and other such methods may optionally beused (optionally also with the above described genetic level fusionmethods), as described for example in U.S. Pat. No. 5,547,853 to Wallneret al, which is hereby incorporated by reference as if fully set forthherein as a non-limiting example only.

According to the present invention, a fusion protein may be preparedfrom a protein of the invention by fusion with a portion of animmunoglobulin comprising a constant region of an immunoglobulin. Morepreferably, the portion of the immunoglobulin comprises a heavy chainconstant region which is optionally and more preferably a human heavychain constant region. The heavy chain constant region is mostpreferably an IgG heavy chain constant region, and optionally and mostpreferably is an Fc chain, most preferably an IgG Fc fragment thatcomprises the hinge, CH2 and CH3 domains. The Fc chain may optionally bea known or “wild type” Fc chain, or alternatively may be mutated ortruncated. The Fc portion of the fusion protein may optionally be variedby isotype or subclass, may be a chimeric or hybrid, and/or may bemodified, for example to improve effector functions, control ofhalf-life, tissue accessibility, augment biophysical characteristicssuch as stability, and improve efficiency of production (and lesscostly). Many modifications useful in construction of disclosed fusionproteins and methods for making them are known in the art, see forexample Mueller, et al, MoI. Immun, 34(6):441-452 (1997), Swann, et al.,Cur. Opin. Immun, 20:493-499 (2008), and Presta, Cur. Opin. Immun.20:460-470 (2008). In some embodiments the Fc region is the native IgG1,IgG2, or IgG4 Fc region. In some embodiments the Fc region is a hybrid,for example a chimeric consisting of IgG2/IgG4 Fc constant regions.

Modifications to the Fc region include, but are not limited to, IgG4modified to prevent binding to Fc gamma receptors and complement, IgG1modified to improve binding to one or more Fc gamma receptors, IgG1modified to minimize effector function (amino acid changes), IgG1 withaltered/no glycan (typically by changing expression host or substitutingthe Asn at position 297), and IgG1 with altered pH-dependent binding toFcRn. The Fc region may include the entire hinge region, or less thanthe entire hinge region.

In another embodiment, the Fc domain may contain one or more amino acidinsertions, deletions or substitutions that reduce binding to the lowaffinity inhibitory Fc receptor CD32B (FcγRIIB) and retain wild-typelevels of binding to or enhance binding to the low affinity activatingFc receptor CD16A (FcγRIIIA)

Another embodiment includes IgG2-4 hybrids and IgG4 mutants that havereduced binding to FcR (Fc receptor) which increase their half life.Representative IgG2-4 hybrids and IgG4 mutants are described in Angal,S. et al., Molecular Immunology, 30(1):105-108 (1993); Mueller, J. etal., Molecular Immunology, 34(6): 441-452 (1997); and U.S. Pat. No.6,982,323 to Wang et al. In some embodiments the IgG1 and/or IgG2 domainis deleted; for example, Angal et al. describe IgG1 and IgG2 havingserine 241 replaced with a proline.

In a further embodiment, the Fc domain contains amino acid insertions,deletions or substitutions that enhance binding to CD16A. A large numberof substitutions in the Fc domain of human IgG1 that increase binding toCD16A and reduce binding to CD32B are known in the art and are describedin Stavenhagen, et al., Cancer Res., 57(18):8882-90 (2007). Exemplaryvariants of human IgG1 Fc domains with reduced binding to CD32B and/orincreased binding to CD16A contain F243L, R929P, Y300L, V305I or P296Lsubstitutions. These amino acid substitutions may be present in a humanIgG1 Fc domain in any combination.

In one embodiment, the human IgG1 Fc domain variant contains a F243L,R929P and Y300L substitution. In another embodiment, the human IgG1 Fcdomain variant contains a F243L, R929P, Y300L, V305I and P296Lsubstitution. In another embodiment, the human IgG1 Fc domain variantcontains an N297A/Q substitution, as these mutations abolishFcγRbinding. Non-limiting, illustrative, exemplary types of mutations aredescribed in US Patent Application No. 20060034852, published on Feb.16, 2006, hereby incorporated by reference as if fully set forth herein.The term “Fc chain” also optionally comprises any type of Fc fragment.

Several of the specific amino acid residues that are important forantibody constant region-mediated activity in the IgG subclass have beenidentified. Inclusion, substitution or exclusion of these specific aminoacids therefore allows for inclusion or exclusion of specificimmunoglobulin constant region-mediated activity. Furthermore, specificchanges may result in aglycosylation for example and/or other desiredchanges to the Fc chain. At least some changes may optionally be made toblock a function of Fc which is considered to be undesirable, such as anundesirable immune system effect, as described in greater detail below.

Non-limiting, illustrative examples of mutations to Fc which may be madeto modulate the activity of the fusion protein include the followingchanges (given with regard to the Fc sequence nomenclature as given byKabat, from Kabat E A et al: Sequences of Proteins of ImmunologicalInterest. US Department of Health and Human Services, NIH, 1991):220C->S; 233-238 ELLGGP->EAEGAP; 265D->A, preferably in combination with434N->A; 297N->A (for example to block N-glycosylation); 318-322EYKCK->AYACA; 330-331AP->SS; or a combination thereof (see for exampleM. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31 for adescription of these mutations and their effect). The construct for theFc chain which features the above changes optionally and preferablycomprises a combination of the hinge region with the CH2 and CH3domains.

The above mutations may optionally be implemented to enhance desiredproperties or alternatively to block non-desired properties. Forexample, aglycosylation of antibodies was shown to maintain the desiredbinding functionality while blocking depletion of T-cells or triggeringcytokine release, which may optionally be undesired functions (see M.Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31).Substitution of 331 proline for serine may block the ability to activatecomplement, which may optionally be considered an undesired function(see M. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31).Changing 330 alanine to serine in combination with this change may alsoenhance the desired effect of blocking the ability to activatecomplement.

Residues 235 and 237 were shown to be involved in antibody-dependentcell-mediated cytotoxicity (ADCC), such that changing the block ofresidues from 233-238 as described may also block such activity if ADCCis considered to be an undesirable function.

Residue 220 is normally a cysteine for Fc from IgG1, which is the siteat which the heavy chain forms a covalent linkage with the light chain.Optionally, this residue may be changed to another amino acid residue(e.g., serine), to avoid any type of covalent linkage (see M. Clark,“Chemical Immunol and Antibody Engineering”, pp 1-31) or by deletion ortruncation.

The above changes to residues 265 and 434 may optionally be implementedto reduce or block binding to the Fc receptor, which may optionallyblock undesired functionality of Fc related to its immune systemfunctions (see “Binding site on Human IgG1 for Fc Receptors”, Shields etal, Vol 276, pp 6591-6604, 2001).

The above changes are intended as illustrations only of optional changesand are not meant to be limiting in any way. Furthermore, the aboveexplanation is provided for descriptive purposes only, without wishingto be bound by a single hypothesis.

In a further embodiment, the fusion protein includes the extracellulardomain of LY6G6F, or a fragment thereof fused to an Ig Fc region.Recombinant IgLY6G6F polypeptides, fragments or fusion proteins thereoffusion proteins can be prepared by fusing the coding region of theextracellular domain of LY6G6F or a fragment thereof to the Fc region ofhuman IgG1 or mouse IgG2a, as described previously (Chapoval, et al.,Methods MoI. Med, 45:247-255 (2000)).

Optionally, LY6G6F ECD refers also to fusion protein, comprising anamino acid sequence of human LY6G6F ECD fused to human immunoglobulinFc. Optionally, said fusion protein comprises the amino acid sequence ofthe human LY6G6F ECD set forth in SEQ ID NO: 2 fused to human IgG1 Fcset forth in any one of SEQ ID NOs:70, 156. Optionally, the amino acidsequence of said fusion protein is set forth in SEQ ID NO:71 or SEQ IDNO:172.

In a further embodiment, the fusion protein includes the extracellulardomain of VSIG10, or a fragment thereof fused to an Ig Fc region.Recombinant IgVSIG10 polypeptides, fragments or fusion proteins thereoffusion proteins can be prepared by fusing the coding region of theextracellular domain of VSIG10 or a fragment thereof to the Fc region ofhuman IgG1 or mouse IgG2a, as described previously (Chapoval, et al.,Methods MoI. Med, 45:247-255 (2000)).

Optionally, VSIG10 ECD refers also to fusion protein, comprising anamino acid sequence of human VSIG10 ECD fused to human immunoglobulinFc. Optionally, said fusion protein comprises the amino acid sequence ofthe human VSIG10 ECD, selected from the amino acid sequences set forthin any one of SEQ ID NOs: 4 and 6, fused to human IgG1 Fc set forth inany one of SEQ ID NOs:70, 156. Optionally, the amino acid sequence ofsaid fusion protein is set forth in any one of SEQ ID NOs:72, 73, 173and 174.

In a further embodiment, the fusion protein includes the extracellulardomain of TMEM25, or a fragment thereof fused to an Ig Fc region.Recombinant IgTMEM25 polypeptides, fragments or fusion proteins thereoffusion proteins can be prepared by fusing the coding region of theextracellular domain of TMEM25 or a fragment thereof to the Fc region ofhuman IgG1 or mouse IgG2a, as described previously (Chapoval, et al.,Methods MoI. Med, 45:247-255 (2000)).

Optionally, TMEM25 ECD refers also to fusion protein, comprising anamino acid sequence of human TMEM25 ECD fused to human immunoglobulinFc. Optionally, said fusion protein comprises the amino acid sequence ofthe human TMEM25 ECD set forth in SEQ ID NO: 8 fused to human IgG1 Fcset forth in any one of SEQ ID NOs:70, 156. Optionally, the amino acidsequence of said fusion protein is set forth in any one of SEQ IDNOs:74, 175.

In a further embodiment, the fusion protein includes the extracellulardomain of LSR, or a fragment thereof fused to an Ig Fc region.Recombinant Ig LSR polypeptides, fragments or fusion proteins thereoffusion proteins can be prepared by fusing the coding region of theextracellular domain of LSR or a fragment thereof to the Fc region ofhuman IgG1 or mouse IgG2a, as described previously (Chapoval, et al.,Methods MoI. Med, 45:247-255. (2000)).

Optionally, LSR ECD refers also to fusion protein, comprising an aminoacid sequence of human LSR ECD fused to human immunoglobulin Fc.Optionally, said fusion protein comprises the amino acid sequence of thehuman LSR ECD, selected from the amino acid sequences set forth in anyone of SEQ ID NOs: 12, 14, 15, 16, 17, 18, 47, 48, 49 and 50, fused tohuman IgG1 Fc set forth in any one of SEQ ID NOs:70, 156. Optionally,the amino acid sequence of said fusion protein is set forth in any oneof SEQ ID NOs:75, 76, 77, 78, 79, 80, 176, 177, 178, 179, 180, and 181.

The aforementioned exemplary fusion proteins can incorporate anycombination of the variants described herein. In another embodiment theterminal lysine of the aforementioned exemplary fusion proteins isdeleted.

The disclosed fusion proteins can be isolated using standard molecularbiology techniques. For example, an expression vector containing a DNAsequence encoding a LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides,fragments or fusion proteins thereof fusion protein is transfected into293 cells by calcium phosphate precipitation and cultured in serum-freeDMEM. The supernatant is collected at 72 h and the fusion protein ispurified by Protein G, or preferably Protein A SEPHAROSE® columns(Pharmacia, Uppsala, Sweden). Optionally, a DNA sequence encoding aLY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, fragments or fusionproteins thereof fusion protein is transfected into GPEx® retrovectorsand expressed in CHO—S cells following four rounds of retrovectortransduction. The protein is clarified from supernatants using protein Achromatography.

In another embodiment the second polypeptide may have a conjugationdomain through which additional molecules can be bound to the LY6G6F,VSIG10, TMEM25 and/or LSR fusion proteins. In one such embodiment, theconjugated molecule is capable of targeting the fusion protein to aparticular organ or tissue; further specific, illustrative, non-limitingexamples of such targeting domains and/or molecules are given below.

In another such embodiment the conjugated molecule is anotherimmunomodulatory agent that can enhance or augment the effects of theLY6G6F, VSIG10, TMEM25 and/or LSR fusion protein. In another embodimentthe conjugated molecule is Polyethylene Glycol (PEG).

Peptide or Polypeptide Linker Domain

The disclosed LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteinsoptionally contain a peptide or polypeptide linker domain that separatesthe LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide from the secondpolypeptide. In one embodiment, the linker domain contains the hingeregion of an immunoglobulin. In a further embodiment, the hinge regionis derived from a human immunoglobulin. Suitable human immunoglobulinsthat the hinge can be derived from include IgG, IgD and IgA. In afurther embodiment, the hinge region is derived from human IgG. Aminoacid sequences of immunoglobulin hinge regions and other domains arewell known in the art. In one embodiment, LY6G6F, VSIG10, TMEM25 and/orLSR fusion polypeptides contain the hinge, CH2 and CH3 regions of ahuman immunoglobulin Cγ1 chain, optionally with the Cys at position 220(according to full length human IgG1, position 5 in SEQ ID NO:70)replaced with a Ser (SEQ ID NO: 156) having at least 85%, 90%, 95%, 99%or 100% sequence homology to amino acid sequence set forth in SEQ IDNO:70:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The hinge can be further shortened to remove amino acids 1, 2, 3, 4, 5,or combinations thereof of any one of SEQ ID NOs: 70 or 156. In oneembodiment, amino acids 1-5 of any one of SEQ ID NOs: 70 or 156 aredeleted. Exemplary LY6G6F, VSIG10, TMEM25 and/or LSR fusion polypeptidescomprised of the hinge, CH2 and CH3 regions of a human immunoglobulinCγ1 chain with the Cys at position 220 replaced with a Ser are set forthin SEQ ID NOs:71, 72, 73, 74, 75, 76, 77, 78, 79, 80.

In another embodiment, LY6G6F, VSIG10, TMEM25 and/or LSR fusionpolypeptides contain the CH2 and CH3 regions of a human immunoglobulinCγ1 chain having at least 85%, 90%, 95%, 99% or 100% sequence homologyto amino acid sequence set forth in SEQ ID NO:157:

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In another embodiment, the LY6G6F, VSIG10, TMEM25 and/or LSR fusionpolypeptides contain the CH2 and CH3 regions of a murine immunoglobulinCγ2a chain at least 85%, 90%, 95%, 99% or 100% sequence homology toamino acid sequence set forth in SEQ ID NO: 158:EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLH NHHTTKSFSRTPGK. Inanother embodiment, the linker domain contains a hinge region of animmunoglobulin as described above, and further includes one or moreadditional immunoglobulin domains.

Other suitable peptide/polypeptide linker domains include naturallyoccurring or non-naturally occurring peptides or polypeptides. Peptidelinker sequences are at least 2 amino acids in length. Optionally thepeptide or polypeptide domains are flexible peptides or polypeptides. A“flexible linker” herein refers to a peptide or polypeptide containingtwo or more amino acid residues joined by peptide bond(s) that providesincreased rotational freedom for two polypeptides linked thereby thanthe two linked polypeptides would have in the absence of the flexiblelinker. Such rotational freedom allows two or more antigen binding sitesjoined by the flexible linker to each access target antigen(s) moreefficiently. Exemplary flexible peptides/polypeptides include, but arenot limited to, the amino acid sequences Gly-Ser (SEQ ID NO:159),Gly-Ser-Gly-Ser (SEQ ID NO:160), Ala-Ser (SEQ ID NO:161),Gly-Gly-Gly-Ser (SEQ ID NO:162), Gly4-Ser (SEQ ID NO:163), (Gly4-Ser)2(SEQ ID NO:164), (Gly4-Ser)3 (SEQ ID NO:165) and (Gly4-Ser)4 (SEQ ID NO:166). Additional flexible peptide/polypeptide sequences are well knownin the art. Other suitable peptide linker domains include helix forminglinkers such as Ala-(Glu-Ala-Ala-Ala-Lys)n-Ala (n=1-5). Additional helixforming peptide/polypeptide sequences are well known in the art.Non-limiting examples of such linkers are depicted in SEQ ID NO:167-171.

Dimerization, Multimerization and Targeting Domains

The fusion proteins disclosed herein optionally contain a dimerizationor multimerization domain that functions to dimerize or multimerize twoor more fusion proteins. The domain that functions to dimerize ormultimerize the fusion proteins can either be a separate domain, oralternatively can be contained within one of the other domains (LY6G6F,VSIG10, TMEM25 and/or LSR polypeptide, second polypeptide, orpeptide/polypeptide linker domain) of the fusion protein.

Dimerization or multinierization can occur between or among two or morefusion proteins through dimerization or multimerization domains.Alternatively, dimerization or multimerization of fusion proteins canoccur by chemical crosslinking. The dimers or multimers that are formedcan be homodimeric/homomultimeric or heterodimeric/heteromultimeric. Thesecond polypeptide “partner” in the LY6G6F, VSIG10, TMEM25 and/or LSRfusion polypeptides may be comprised of one or more other proteins,protein fragments or peptides as described herein, including but notlimited to any immunoglobulin (Ig) protein or portion thereof,preferably the Fc region, or a portion of a biologically or chemicallyactive protein such as the papillomavirus E7 gene product,melanoma-associated antigen p97), and HIV env protein (gp120). The“partner” is optionally selected to provide a soluble dimer/multimerand/or for one or more other biological activities as described herein.

A “dimerization domain” is formed by the association of at least twoamino acid residues or of at least two peptides or polypeptides (whichmay have the same, or different, amino acid sequences). The peptides orpolypeptides may interact with each other through covalent and/ornon-covalent associations). Optional dimerization domains contain atleast one cysteine that is capable of forming an intermoleculardisulfide bond with a cysteine on the partner fusion protein. Thedimerization domain can contain one or more cysteine residues such thatdisulfide bond(s) can form between the partner fusion proteins. In oneembodiment, dimerization domains contain one, two or three to about tencysteine residues. In a further embodiment, the dimerization domain isthe hinge region of an immunoglobulin.

Additional exemplary dimerization domains can be any known in the artand include, but not limited to, coiled coils, acid patches, zincfingers, calcium hands, a C_(H)1-C_(L) pair, an “interface” with anengineered “knob” and/or “protruberance” as described in U.S. Pat. No.5,821,333, leucine zippers (e.g., from jun and/or fos) (U.S. Pat. No.5,932,448), and/or the yeast transcriptional activator GCN4, SH2 (srchomology 2), SH3 (src Homology 3) (Vidal, et al, Biochemistry, 43,7336-44 ((2004)), phosphotyrosine binding (PTB) (Zhou, et al., Nature,378:584-592 (1995)), WW (Sudol, Prog, Biochys. MoL Bio., 65:113-132(1996)), PDZ (Kim, et al., Nature, 378: 85-88 (1995); Komau, et al,Science, 269.1737-1740 (1995)) 14-3-3, WD40 (Hu5 et al., J Biol Chem.,273, 33489-33494 (1998)) EH, Lim, an isoleucine zipper, a receptor dimerpair (e.g., interleukin-8 receptor (IL-8R); and integrin heterodimerssuch as LFA-I and GPIIIb/IIIa), or the dimerization region(s) thereof,dimeric ligand polypeptides (e.g. nerve growth factor (NGF),neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growthfactor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derivedneurotrophic factor (BDNF) (Arakawa, et al., J Biol. Chem., 269(45):27833-27839 (1994) and Radziejewski, et al., Biochem., 32(48): 1350(1993)) and can also be variants of these domains in which the affinityis altered. The polypeptide pairs can be identified by methods known inthe art, including yeast two hybrid screens. Yeast two hybrid screensare described in U.S. Pat. Nos. 5,283,173 and 6,562,576. Affinitiesbetween a pair of interacting domains can be determined using methodsknown in the art, including as described in Katahira, et at, J. BiolChem, 277, 9242-9246 (2002)). Alternatively, a library of peptidesequences can be screened for heterodimerization, for example, using themethods described in WO 01/00814. Useful methods for protein-proteininteractions are also described in U.S. Pat. No. 6,790,624.

A “multimerization domain” is a domain that causes three or morepeptides or polypeptides to interact with each other through covalentand/or non-covalent association(s). Suitable multimerization domainsinclude, but are not limited to, coiled-coil domains. A coiled-coil is apeptide sequence with a contiguous pattern of mainly hydrophobicresidues spaced 3 and 4 residues apart, usually in a sequence of sevenamino acids (heptad repeat) or eleven amino acids (undecad repeat),which assembles (folds) to form a multimeric bundle of helices.Coiled-coils with sequences including some irregular distribution of the3 and 4 residues spacing are also contemplated. Hydrophobic residues arein particular the hydrophobic amino acids Val, Ile, Leu, Met, Tyr, Pheand Trp. “Mainly hydrophobic” means that at least 50% of the residuesmust be selected from the mentioned hydrophobic amino acids.

The coiled coil domain may be derived from laminin In the extracellularspace, the heterotrimeric coiled coil protein laminin plays an importantrole in the formation of basement membranes. Apparently, themultifunctional oligomeric structure is required for laminin function.Coiled coil domains may also be derived from the thrombospondins inwhich three (TSP-I and TSP-2) or five (TSP-3, TSP-4 and TSP-5) chainsare connected, or from COMP (COMPcc) (Guo, et at., EMBO J, 1998, 17:5265-5272) which folds into a parallel five-stranded coiled coil(Malashkevich, et al., Science, 274: 761-765 (1996)).

Additional non limiting examples of coiled-coil domains derived fromother proteins, and other domains that mediate polypeptidemultimerization are known in the art such as the vasodialator-stimulatedphosphoprotein (VASP) domain, matrilin-1 (CMP), viral fusion peptides,soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Proteinreceptor (SNARE) complexes, leucine-rich repeats, certain tRNAsynthetases, are suitable for use in the disclosed fusion proteins.

In another embodiment, LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides,fusion proteins, or fragments thereof can be induced to form multimersby binding to a second multivalent polypeptide, such as an antibody.Antibodies suitable for use to multimerize LY6G6F, VSIG10, TMEM25 and/orLSR polypeptides, fusion proteins, or fragments thereof include, but arenot limited to, IgM antibodies and cross-linked, multivalent IgG, IgA,IgD, or IgE complexes.

Dimerization or multimerization can occur between or among two or morefusion proteins through dimerization or multimerization domains,including those described above. Alternatively, dimerization ormultimerization of fusion proteins can occur by chemical crosslinking.Fusion protein dimers can be homodimers or heterodimers. Fusion proteinmultimers can be homomultimers or heteromultimers. Fusion protein dimersas disclosed herein are of formula II:

N-R1-R2-R3-C

N-R4-R5-R6-C or, alternatively, are of formula

N-R1-R2-R3-C

C-R4-R5-R6-N

wherein the fusion proteins of the dimer provided by formula II aredefined as being in a parallel orientation and the fusion proteins ofthe dimer provided by formula III are defined as being in anantiparallel orientation. Parallel and antiparallel dimers are alsoreferred to as cis and trans dimers, respectively. “N” and “C” representthe N- and C-termini of the fusion protein, respectively. The fusionprotein constituents “R1”, “R2” and “R3” are as defined above withrespect to formula I. With respect to both formula II and formula III,“R4” is a LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide or a secondpolypeptide, “R5” is an optional peptide/polypeptide linker domain, and“R6” is a LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide or a secondpolypeptide, wherein “R6” is a LY6G6F, VSIG10, TMEM25 and/or LSRpolypeptide when “R4” is a second polypeptide, and “R6′” is a secondpolypeptide when “R4” is a LY6G6F, VSIG10, TMEM25 and/or LSRpolypeptide. In one embodiment, “R1” is a LY6G6F, VSIG10, TMEM25 and/orLSR polypeptide, “R4” is also a LY6G6F, VSIG10, TMEM25 and/or LSRpolypeptide, and “R3” and “R6” are both second polypeptides.

Fusion protein dimers of formula II are defined as homodimers when“R1”=“R4”, “R2”=“R5” and “R3”=“R6”. Similarly, fusion protein dimers offormula III are defined as homodimers when “R1”=“R6”, “R2”=“R5” and“R3”=“R4”. Fusion protein dimers are defined as heterodimers when theseconditions are not met for any reason. For example, heterodimers maycontain domain orientations that meet these conditions (i.e., for adimer according to formula II, “R1” and “R4” are both LY6G6F, VSIG10,TMEM25 and/or LSR polypeptides, “R2” and “R5” are bothpeptide/polypeptide linker domains and “R3” and “R6” are both secondpolypeptides), however the species of one or more of these domains isnot identical. For example, although “R3” and “R6” may both be LY6G6F,VSIG10, TMEM25 and/or LSR polypeptides, one polypeptide may contain awild-type LY6G6F, VSIG10, TMEM25 and/or LSR amino acid sequence whilethe other polypeptide may be a variant LY6G6F, VSIG10, TMEM25 and/or LSRpolypeptide. An exemplary variant LY6G6F, VSIG10, TMEM25 and/or LSRpolypeptide is LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide that hasbeen modified to have increased or decreased binding to a target cell,increased activity on immune cells, increased or decreased half life orstability. Dimers of fusion proteins that contain either a CHI or CLregion of an immunoglobulin as part of the polypeptide linker domainpreferably form heterodimers wherein one fusion protein of the dimercontains a CHI region and the other fusion protein of the dimer containsa CL region.

Fusion proteins can also be used to form multimers. As with dimers,multimers may be parallel multimers, in which all fusion proteins of themultimer are aligned in the same orientation with respect to their N-and C-termini. Multimers may be antiparallel multimers, in which thefusion proteins of the multimer are alternatively aligned in oppositeorientations with respect to their N- and C-termini. Multimers (parallelor antiparallel) can be either homomultimers or heteromultimers. Thefusion protein is optionally produced in dimeric form; more preferably,the fusion is performed at the genetic level as described below, byjoining polynucleotide sequences corresponding to the two (or more)proteins, portions of proteins and/or peptides, such that a joined orfused protein is produced by a cell according to the joinedpolynucleotide sequence. A description of preparation for such fusionproteins is described with regard to U.S. Pat. No. 5,851,795 to Linsleyet al, which is hereby incorporated by reference as if fully set forthherein as a non-limiting example only.

Targeting Domains

The LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides and fusion proteinscan contain a targeting domain to target the molecule to specific sitesin the body. Optional targeting domains target the molecule to areas ofinflammation. Exemplary targeting domains are antibodies, or antigenbinding fragments thereof that are specific for inflamed tissue or to aproinflammatory cytokine including but not limited to IL17, IL-4, IL-6,IL-12, IL-21, IL-22, and IL-23. In the case of neurological disorderssuch as Multiple Sclerosis, the targeting domain may target the moleculeto the CNS or may bind to VCAM-I on the vascular epithelium. Additionaltargeting domains can be peptide aptamers specific for a proinflammatorymolecule. In other embodiments, the LY6G6F, VSIG10, TMEM25 and/or LSRfusion protein can include a binding partner specific for a polypeptidedisplayed on the surface of an immune cell, for example a T cell. Instill other embodiments, the targeting domain specifically targetsactivated immune cells. Optional immune cells that are targeted includeTh0, Th1, Th 17, Th2 and Th22 T cells, other cells that secrete, orcause other cells to secrete inflammatory molecules including, but notlimited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6,IL-23, IL-22, IL-21, and MMPs, and Tregs. For example, a targetingdomain for Tregs may bind specifically to CD25. The above changes areintended as illustrations only of optional changes and are not meant tobe limiting in any way. Furthermore, the above explanation is providedfor descriptive purposes only, without wishing to be bound by a singlehypothesis.

Addition of Groups

If a protein according to the present invention is a linear molecule, itis possible to place various functional groups at various points on thelinear molecule which are susceptible to or suitable for chemicalmodification. Functional groups can be added to the termini of linearforms of the protein according to at least some embodiments of theinvention. In some embodiments, the functional groups improve theactivity of the protein with regard to one or more characteristics,including but not limited to, improvement in stability, penetration(through cellular membranes and/or tissue barriers), tissuelocalization, efficacy, decreased clearance, decreased toxicity,improved selectivity, improved resistance to expulsion by cellularpumps, and the like. For convenience sake and without wishing to belimiting, the free N-terminus of one of the sequences contained in thecompositions according to at least some embodiments of the inventionwill be termed as the N-terminus of the composition, and the freeC-terminal of the sequence will be considered as the C-terminus of thecomposition. Either the C-terminus or the N-terminus of the sequences,or both, can be linked to a carboxylic acid functional groups or anamine functional group, respectively.

Non-limiting examples of suitable functional groups are described inGreen and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley andSons, Chapters 5 and 7, 1991, the teachings of which are incorporatedherein by reference. Preferred protecting groups are those thatfacilitate transport of the active ingredient attached thereto into acell, for example, by reducing the hydrophilicity and increasing thelipophilicity of the active ingredient, these being an example for “amoiety for transport across cellular membranes”.

These moieties can optionally and preferably be cleaved in vivo, eitherby hydrolysis or enzymatically, inside the cell. (Ditter et al., J.Pharm. Sci. 57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828 (1968);Ditter et al., J. Pharm. Sci. 58:557 (1969); King et al., Biochemistry26:2294 (1987); Lindberg et al., Drug Metabolism and Disposition 17:311(1989); and Tunek et al., Biochem. Pharm. 37:3867 (1988), Anderson etal., Arch. Biochem. Biophys. 239:538 (1985) and Singhal et al., FASEB J.1:220 (1987)). Hydroxyl protecting groups include esters, carbonates andcarbamate protecting groups Amine protecting groups include alkoxy andaryloxy carbonyl groups, as described above for N-terminal protectinggroups. Carboxylic acid protecting groups include aliphatic, benzylicand aryl esters, as described above for C-terminal protecting groups. Inone embodiment, the carboxylic acid group in the side chain of one ormore glutamic acid or aspartic acid residue in a composition of thepresent invention is protected, preferably with a methyl, ethyl, benzylor substituted benzyl ester, more preferably as a benzyl ester.

Non-limiting, illustrative examples of N-terminal protecting groupsinclude acyl groups (—CO—R1) and alkoxy carbonyl or aryloxy carbonylgroups (—CO—O—R1), wherein R1 is an aliphatic, substituted aliphatic,benzyl, substituted benzyl, aromatic or a substituted aromatic group.Specific examples of acyl groups include but are not limited to acetyl,(ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—, n-butyl-CO—, sec-butyl-CO—,t-butyl-CO—, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoylphenyl-CO—, substituted phenyl-CO—, benzyl-CO— and (substitutedbenzyl)-CO—. Examples of alkoxy carbonyl and aryloxy carbonyl groupsinclude CH3-O—CO—, (ethyl)-O—CO—, n-propyl-O—CO—, iso-propyl-O—CO—,n-butyl-O—CO—, sec-butyl-O—CO—, t-butyl-O—CO—, phenyl-O— CO—,substituted phenyl-O—CO— and benzyl-O—CO—, (substituted benzyl)-O—CO—,Adamantan, naphtalen, myristoleyl, toluen, biphenyl, cinnamoyl,nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane, norbornane, orZ-caproic. In order to facilitate the N-acylation, one to four glycineresidues can be present in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected,for example, by a group including but not limited to an amide (i.e., thehydroxyl group at the C-terminus is replaced with —NH₂, —NHR₂ and—NR₂R₃) or ester (i.e. the hydroxyl group at the C-terminus is replacedwith —OR₂). R₂ and R₃ are optionally independently an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aryl or a substitutedaryl group. In addition, taken together with the nitrogen atom, R₂ andR₃ can optionally form a C4 to C8 heterocyclic ring with from about 0-2additional heteroatoms such as nitrogen, oxygen or sulfur. Non-limitingsuitable examples of suitable heterocyclic rings include piperidinyl,pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples ofC-terminal protecting groups include but are not limited to —NH₂,—NHCH₃, —N(CH₃)₂, —NH(ethyl), —N(ethyl)₂, —N(methyl) (ethyl),—NH(benzyl), —N(C1-C4 alkyl)(benzyl), —NH(phenyl), —N(C1-C4 alkyl)(phenyl), —OCH₃, —O-(ethyl), —O-(n-propyl), —O-(n-butyl),—O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benzyl and —O-phenyl.

Substitution by Peptidomimetic Moieties

A “peptidomimetic organic moiety” can optionally be substituted foramino acid residues in the composition of this invention both asconservative and as non-conservative substitutions. These moieties arealso termed “non-natural amino acids” and may optionally replace aminoacid residues, amino acids or act as spacer groups within the peptidesin lieu of deleted amino acids. The peptidomimetic organic moietiesoptionally and preferably have steric, electronic or configurationalproperties similar to the replaced amino acid and such peptidomimeticsare used to replace amino acids in the essential positions, and areconsidered conservative substitutions. However such similarities are notnecessarily required. According to preferred embodiments of the presentinvention, one or more peptidomimetics are selected such that thecomposition at least substantially retains its physiological activity ascompared to the native protein according to the present invention.

Peptidomimetics may optionally be used to inhibit degradation of thepeptides by enzymatic or other degradative processes. Thepeptidomimetics can optionally and preferably be produced by organicsynthetic techniques. Non-limiting examples of suitable peptidomimeticsinclude D amino acids of the corresponding L amino acids, tetrazol(Zabrocki et al., J. Am. Chem. Soc. 110:5875-5880 (1988)); isosteres ofamide bonds (Jones et al., Tetrahedron Lett. 29: 3853-3856 (1988));LL-3-amino-2-propenidone-6-carboxylic acid (LL-Acp) (Kemp et al., J.Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in Kemp etal., Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp et al.,Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al., Tetrahedron Lett.29:4935-4938 (1988) and Kemp et al., J. Org. Chem. 54:109-115 (1987).Other suitable but exemplary peptidomimetics are shown in Nagai andSato, Tetrahedron Lett. 26:647-650 (1985); Di Maio et al., J. Chem. Soc.Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317(1989); Olson et al., J. Am. Chem. Soc. 112:323-333 (1990); Garvey etal., J. Org. Chem. 56:436 (1990). Further suitable exemplarypeptidomimetics includehydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., J.Takeda Res. Labs 43:53-76 (1989));1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (Kazmierski et al., J. Am.Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone carboxylic acid(HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)); (2S,3S)-methyl-phenylalanine, (2S, 3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R, 3R)-methyl-phenylalanine (Kazmierskiand Hruby, Tetrahedron Lett. (1991)).

Exemplary, illustrative but non-limiting non-natural amino acids includebeta-amino acids (beta3 and beta2), homo-amino acids, cyclic aminoacids, aromatic amino acids, Pro and Pyr derivatives, 3-substitutedAlanine derivatives, Glycine derivatives, ring-substituted Phe and TyrDerivatives, linear core amino acids or diamino acids. They areavailable from a variety of suppliers, such as Sigma-Aldrich (USA) forexample.

Protein Chemical Modifications

In the present invention any part of a protein according to at leastsome embodiments of the invention may optionally be chemically modified,i.e. changed by addition of functional groups. For example the sideamino acid residues appearing in the native sequence may optionally bemodified, although as described below alternatively other parts of theprotein may optionally be modified, in addition to or in place of theside amino acid residues. The modification may optionally be performedduring synthesis of the molecule if a chemical synthetic process isfollowed, for example by adding a chemically modified amino acid.However, chemical modification of an amino acid when it is alreadypresent in the molecule (“in situ” modification) is also possible.

The amino acid of any of the sequence regions of the molecule canoptionally be modified according to any one of the following exemplarytypes of modification (in the peptide conceptually viewed as “chemicallymodified”). Non-limiting exemplary types of modification includecarboxymethylation, acylation, phosphorylation, glycosylation or fattyacylation. Ether bonds can optionally be used to join the serine orthreonine hydroxyl to the hydroxyl of a sugar. Amide bonds canoptionally be used to join the glutamate or aspartate carboxyl groups toan amino group on a sugar (Garg and Jeanloz, Advances in CarbohydrateChemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang.Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds canalso optionally be formed between amino acids and carbohydrates. Fattyacid acyl derivatives can optionally be made, for example, by acylationof a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry,Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden,1078-1079 (1990)).

As used herein the term “chemical modification”, when referring to aprotein or peptide according to the present invention, refers to aprotein or peptide where at least one of its amino acid residues ismodified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art. Examples of the numerous knownmodifications typically include, but are not limited to: acetylation,acylation, amidation, ADP-ribosylation, glycosylation, GPI anchorformation, covalent attachment of a lipid or lipid derivative,methylation, myristylation, pegylation, prenylation, phosphorylation,ubiquitination, or any similar process.

Other types of modifications optionally include the addition of acycloalkane moiety to a biological molecule, such as a protein, asdescribed in PCT Application No. WO 2006/050262, hereby incorporated byreference as if fully set forth herein. These moieties are designed foruse with biomolecules and may optionally be used to impart variousproperties to proteins.

Furthermore, optionally any point on a protein may be modified. Forexample, pegylation of a glycosylation moiety on a protein mayoptionally be performed, as described in PCT Application No. WO2006/050247, hereby incorporated by reference as if fully set forthherein. One or more polyethylene glycol (PEG) groups may optionally beadded to O-linked and/or N-linked glycosylation. The PEG group mayoptionally be branched or linear. Optionally any type of water-solublepolymer may be attached to a glycosylation site on a protein through aglycosyl linker.

Altered Glycosylation

Proteins according to at least some embodiments of the invention may bemodified to have an altered glycosylation pattern (i.e., altered fromthe original or native glycosylation pattern). As used herein, “altered”means having one or more carbohydrate moieties deleted, and/or having atleast one glycosylation site added to the original protein.

Glycosylation of proteins is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequences,asparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to proteins according to at least someembodiments of the invention is conveniently accomplished by alteringthe amino acid sequence of the protein such that it contains one or moreof the above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues in thesequence of the original protein (for O-linked glycosylation sites). Theprotein's amino acid sequence may also be altered by introducing changesat the DNA level.

Another means of increasing the number of carbohydrate moieties onproteins is by chemical or enzymatic coupling of glycosides to the aminoacid residues of the protein. Depending on the coupling mode used, thesugars may be attached to (a) arginine and histidine, (b) free carboxylgroups, (c) free sulfhydryl groups such as those of cysteine, (d) freehydroxyl groups such as those of serine, threonine, or hydroxyproline,(e) aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev.Biochem., 22: 259-306 (1981).

Removal of any carbohydrate moieties present on proteins according to atleast some embodiments of the invention may be accomplished chemicallyor enzymatically. Chemical deglycosylation requires exposure of theprotein to trifluoromethanesulfonic acid, or an equivalent compound.This treatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), leavingthe amino acid sequence intact.

Chemical deglycosylation is described by Hakimuddin et al., Arch.Biochem. Biophys., 259: 52 (1987); and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on proteins canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138: 350 (1987).

Methods of Use

As used herein “therapeutic agent” is any one of the LY6G6F, VSIG10,TMEM25 and/or LSR proteins and polypeptides according to at least someembodiments of the present invention, or orthologs, or fragmentsthereof, especially the ectodomain or secreted forms of LY6G6F, VSIG10,TMEM25 and/or LSR proteins, and/or fusion protein, and/or multimericprotein containing same, or nucleic acid sequence or fragments thereofof LY6G6F, VSIG10, TMEM25 and/or LSR, as well as drugs whichspecifically bind to the LY6G6F, VSIG10, TMEM25 and/or LSR proteins,and/or drugs which agonize or antagonize the binding of other moietiesto the LY6G6F, VSIG10, TMEM25 and/or LSR proteins, and/or drugs whichmodulate (agonize or antagonize) at least one LY6G6F, VSIG10, TMEM25and/or LSR related biological activity. Such drugs include monoclonaland/or polyclonal antibodies, and/or antigen binding fragments, and/orconjugates containing same, and/or alternative scaffolds, thereofcomprising an antigen binding site that binds specifically to any one ofthe LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides or an epitopethereof. Such drugs by way of example also include small molecules,peptides, ribozymes, aptamers, antisense molecules, siRNA's and thelike.

Stimulation of activity of LY6G6F, VSIG10, TMEM25 and/or LSR isdesirable in situations in which LY6G6F, VSIG10, TMEM25 and/or LSR isabnormally downregulated, and/or situations in which increased activityof LY6G6F, VSIG10, TMEM25 and/or LSR is likely to have a beneficialeffect. Likewise, inhibition of activity of LY6G6F, VSIG10, TMEM25and/or LSR is desirable in situations in which LY6G6F, VSIG10, TMEM25and/or LSR is abnormally upregulated, and/or situations in whichdecreased activity of LY6G6F, VSIG10, TMEM25 and/or LSR is likely tohave beneficial effect.

As mentioned herein above, the therapeutic agents can be used to treatimmune related disorders as recited herein, and/or autoimmune disordersas recited herein, and/or infectious disorders as recited herein, and/orcancer as recited herein and/or for blocking and/or promoting immunecostimulation mediated by any one of the LY6G6F, VSIG10, TMEM25 and/orLSR polypeptides.

According to an additional aspect of the present invention thetherapeutic agents can be used to prevent pathologic inhibition of Tcell activity, such as that directed against cancer cells or chronicinfections; and/or prevent pathologic stimulation of T cell activity,such as that directed against autoantigens in autoimmune diseases. Forexample, these molecules can be administered to cells in culture, invitro or ex vivo, or to human subjects, e.g., in vivo, to treat, preventand to diagnose a variety of disorders. Preferred subjects include humanpatients, having disorders mediated by cells expressing the LY6G6F,VSIG10, TMEM25 and/or LSR protein, and cells that possess LY6G6F,VSIG10, TMEM25 and/or LSR activity.

According to an additional aspect of the present invention thetherapeutic agents can be used to inhibit T cell activation, as can bemanifested for example by T cell proliferation and cytokine secretion.

According to an additional aspect of the present invention thetherapeutic agents can be used to elicit in vivo or in vitro one or moreof the following biological activities: to inhibit the growth of and/orkill a cell expressing LY6G6F, VSIG10, TMEM25 and/or LSR; to mediatephagocytosis or ADCC of a cell expressing LY6G6F, VSIG10, TMEM25 and/orLSR in the presence of human effector cells, or to block LY6G6F, VSIG10,TMEM25 and/or LSR ligand binding to LY6G6F, VSIG10, TMEM25 and/or LSR,respectively.

Thus, according to an additional aspect of the present invention thereis provided a method of treating immune related disorders as recitedherein, and/or autoimmune disorders as recited herein, and/or infectiousdisorders as recited herein, and/or cancer as recited herein, and/or forblocking or promoting immune stimulation mediated by the LY6G6F, VSIG10,TMEM25 and/or LSR polypeptide in a subject by administering to a subjectin need thereof an effective amount of any one of the therapeutic agentsand/or a pharmaceutical composition comprising any of the therapeuticagents and further comprising a pharmaceutically acceptable diluent orcarrier.

The subject according to the present invention is a mammal, preferably ahuman which is diagnosed with one of the disease, disorder or conditionsdescribed hereinabove, or alternatively is predisposed to at least onetype of cancer and/or infectious disorders, and/or immune relateddisorder.

As used herein the term “treating” refers to preventing, delaying theonset of, curing, reversing, attenuating, alleviating, minimizing,suppressing or halting the deleterious effects of the above-describeddiseases, disorders or conditions. It also includes managing the diseaseas described above. By “manage” it is meant reducing the severity of thedisease, reducing the frequency of episodes of the disease, reducing theduration of such episodes, reducing the severity of such episodes andthe like.

Treating, according to the present invention, can be effected byspecifically upregulating the expression of at least one of thepolypeptides of the present invention in the subject.

It will be appreciated that treatment of the above-described diseasesaccording to the present invention may be combined with other treatmentmethods known in the art (i.e., combination therapy). Thus thetherapeutic agents and/or a pharmaceutical composition comprising same,as recited herein, according to at least some embodiments of the presentinvention can also be used in combination with one or more of thefollowing agents to regulate an immune response: soluble gp39 (alsoknown as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD29, solubleCD40, soluble CD80 (e.g. ATCC 68627), soluble CD86, soluble CD28 (e.g.68628), soluble CD56, soluble Thy-1, soluble CD3, soluble TCR, solubleVLA-4, soluble VCAM-1, soluble LECAM-1, soluble ELAM-1, soluble CD44,antibodies reactive with gp39 (e.g. ATCC HB-10916, ATCC HB-12055 andATCC HB-12056), antibodies reactive with CD40 (e.g. ATCC HB-9110),antibodies reactive with B7 (e.g. ATCC HB-253, ATCC CRL-2223, ATCCCRL-2226, ATCC HB-301, ATCC HB-11341, etc), antibodies reactive withCD28 (e.g. ATCC HB-11944 or mAb 9.3), antibodies reactive with LFA-1(e.g. ATCC HB-9579 and ATCC TIB-213), antibodies reactive with LFA-2,antibodies reactive with IL-2, antibodies reactive with IL-12,antibodies reactive with IFN-gamma, antibodies reactive with CD2,antibodies reactive with CD48, antibodies reactive with any ICAM (e.g.,ICAM-1 (ATCC CRL-2252), ICAM-2 and ICAM-3), antibodies reactive withCTLA4 (e.g. ATCC HB-304), antibodies reactive with Thy-1, antibodiesreactive with CD56, antibodies reactive with CD3, antibodies reactivewith CD29, antibodies reactive with TCR, antibodies reactive with VLA-4,antibodies reactive with VCAM-1, antibodies reactive with LECAM-1,antibodies reactive with ELAM-1, antibodies reactive with CD44;L104EA29YIg, CD80 monoclonal antibodies (mAbs), CD86 mAbs, gp39 mAbs,CD40 mAbs, CD28 mAbs; anti-LFA1 mAbs, antibodies or other agentstargeting mechanisms of the immune system such as CD52 (alemtuzumab),CD25 (daclizumab), VLA-4 (natalizumab), CD20 (rituximab), IL2R(daclizumab) and MS4A1 (ocrelizumab); novel oral immunomodulating agentshave shown to prevent lymphocyte recirculation from lymphoid organs suchas fingolimod (FTY720) or leading to lymphocyte depletion such asmylinax (oral cladribine) or teriflunomide; and agents that preventimmunoactivation such as panaclar (dimethyl fumarate BG-12) orlaquinimod (ABR216062). Other combinations will be readily appreciatedand understood by persons skilled in the art. In some embodiments, thetherapeutic agents can be used to attenuate or reverse the activity of apro-inflammatory drug, and/or limit the adverse effects of such drugs.

As persons skilled in the art will readily understand, the combinationcan include the therapeutic agents and/or a pharmaceutical compositioncomprising same, according to at least some embodiments of the inventionand one other immunosuppressive agent; the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, with twoother immunosuppressive agents, the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, withthree other immunosuppressive agents, etc. The determination of theoptimal combination and dosages can be determined and optimized usingmethods well known in the art.

The therapeutic agent according to the present invention and one or moreother therapeutic agents can be administered in either order orsimultaneously. The other therapeutic agents are for example, acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Thecomposition can be linked to the agent (as an immunocomplex) or can beadministered separately from the agent. In the latter case (separateadministration), the composition can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, areonly effective at levels which are toxic or subtoxic to a patient.Cisplatin is intravenously administered as a 100 mg/dose once every fourweeks and adriamycin is intravenously administered as a 60-75 mg/ml doseonce every 21 days.

Co-administration of the human anti-LY6G6F, anti-VSIG10, anti-TMEM25and/or anti-LSR antibodies, or antigen binding fragments thereof,according to at least some embodiments of the present invention withchemotherapeutic agents provides two anti-cancer agents which operatevia different mechanisms which yield a cytotoxic effect to human tumorcells. Such co-administration can solve problems due to development ofresistance to drugs or a change in the antigenicity of the tumor cellswhich would render them unreactive with the antibody. Target-specificeffector cells, e.g., effector cells linked to compositions (e.g., humanantibodies, multispecific and bispecific molecules) according to atleast some embodiments of the invention can also be used as therapeuticagents. Effector cells for targeting can be human leukocytes such asmacrophages, neutrophils or monocytes. Other cells include eosinophils,natural killer cells and other IgG- or IgA-receptor bearing cells. Ifdesired, effector cells can be obtained from the subject to be treated.The target-specific effector cells can be administered as a suspensionof cells in a physiologically acceptable solution. The number of cellsadministered can be in the order of 10-8 to 10-9 but will vary dependingon the therapeutic purpose. In general, the amount will be sufficient toobtain localization at the target cell, e.g., a tumor cell expressingLY6G6F, VSIG10, TMEM25 and/or LSR proteins, and to effect cell killingby, e.g., phagocytosis. Routes of administration can also vary.

Therapy with target-specific effector cells can be performed inconjunction with other techniques for removal of targeted cells. Forexample, anti-tumor therapy using the compositions (e.g., humanantibodies, multispecific and bispecific molecules) according to atleast some embodiments of the invention and/or effector cells armed withthese compositions can be used in conjunction with chemotherapy.Additionally, combination immunotherapy may be used to direct twodistinct cytotoxic effector populations toward tumor cell rejection. Forexample, anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSRantibodies linked to anti-Fc-gamma RI or anti-CD3 may be used inconjunction with IgG- or IgA-receptor specific binding agents.

Bispecific and multispecific molecules according to at least someembodiments of the invention can also be used to modulate FcgammaR orFcgammaR levels on effector cells, such as by capping and elimination ofreceptors on the cell surface. Mixtures of anti-Fc receptors can also beused for this purpose.

The invention also encompasses the use of the compositions according toat least some embodiments of the invention in combination with otherpharmaceutical agents to treat immune system diseases. For example,autoimmune disease may be treated with molecules according to at leastsome embodiments of the invention in conjunction with, but not limitedto, immunosuppressants such as corticosteroids, cyclosporin,cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin,tacrolimus, biological agents such as TNF-alpha blockers or antagonists,or any other biological agent targeting any inflammatory cytokine,nonsteroidal antiinflammatory drugs/Cox-2 inhibitors,hydroxychloroquine, sulphasalazopryine, gold salts, etanercept,infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab,cytoxan, interferon beta-1a, interferon beta-1b, glatiramer acetate,mitoxantrone hydrochloride, anakinra and/or other biologics and/orintravenous immunoglobulin (IVIG). Non-limiting examples of such knowntherapeutics include interferons, such as IFN-beta-1a (REBIF®. AVONEX®and CINNOVEX®) and IFN-beta-1b (BETASERON®, EXTAVIA®, BETAFERON®,ZIFERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab(TYSABRI®); and mitoxantrone (NOVANTRONE®), a cytotoxic agent.

Thus, treatment of multiple sclerosis using the agents according to atleast some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treating multiplesclerosis. Non-limiting examples of such known therapeutic agent ormethod for treating multiple sclerosis include interferon class,IFN-beta-1a (REBIF®. AVONEX® and CINNOVEX®) and IFN-beta-1b (BETASERON®,EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), apolypeptide; natalizumab (TYSABRI®); and mitoxantrone (NOVANTRONE®), acytotoxic agent, Fampridine (AMPYRA®). Other drugs includecorticosteroids, methotrexate, cyclophosphamide, azathioprine, andintravenous immunoglobulin (IVIG), inosine, Ocrelizumab (R1594), Mylinax(Caldribine), alemtuzumab (Campath), daclizumab (Zenapax),Panaclar/dimethyl fumarate (BG-12), Teriflunomide (HMR1726), fingolimod(FTY720), laquinimod (ABR216062), as well as Haematopoietic stem celltransplantation, Neurovax, Rituximab (Rituxan) BCG vaccine, low dosenaltrexone, helminthic therapy, angioplasty, venous stents, andalternative therapy, such as vitamin D, polyunsaturated fats, medicalmarijuana.

Thus, treatment of rheumatoid arthritis, using the agents according toat least some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treatingrheumatoid arthritis. Non-limiting examples of such known therapeuticagents or methods for treating rheumatoid arthritis includeglucocorticoids, nonsteroidal anti-inflammatory drug (NSAID) such assalicylates, or cyclooxygenase-2 inhibitors, ibuprofen and naproxen,diclofenac, indomethacin, etodolac Disease-modifying antirheumatic drugs(DMARDs)—Oral DMARDs: Auranofin (Ridaura), Azathioprine (Imuran),Cyclosporine (Sandimmune, Gengraf, Neoral, generic), D-Penicillamine(Cuprimine), Hydroxychloroquine (Plaquenil), IM gold Gold sodiumthiomalate (Myochrysine) Aurothioglucose (Solganal), Leflunomide(Arava), Methotrexate (Rheumatrex), Minocycline (Minocin),Staphylococcal protein A immunoadsorption (Prosorba column),Sulfasalazine (Azulfidine). Biologic DMARDs: TNF-α blockers includingAdalimumab (Humira), Etanercept (Enbrel), Infliximab (Remicade),golimumab (Simponi), certolizumab pegol (Cimzia), and other BiologicalDMARDs, such as Anakinra (Kineret), Rituximab (Rituxan), Tocilizumab(Actemra), CD28 inhibitor including Abatacept (Orencia) and Belatacept.

Thus, treatment of IBD, using the agents according to at least someembodiments of the present invention may be combined with, for example,any known therapeutic agent or method for treating IBD. Non-limitingexamples of such known therapeutic agents or methods for treating IBDinclude immunosuppression to control the symptom, such as prednisone,Mesalazine (including Asacol, Pentasa, Lialda, Aspiro), azathioprine(Imuran), methotrexate, or 6-mercaptopurine, steroids, Ondansetron,TNF-α blockers (including infliximab, adalimumab golimumab, certolizumabpegol), Orencia (abatacept), ustekinumab (Stelara®), Briakinumab(ABT-874), Certolizumab pegol (Cimzia®), ITF2357 (givinostat),Natalizumab (Tysabri), Firategrast (SB-683699), Remicade (infliximab),vedolizumab (MLN0002), other drugs including GSK1605786 CCX282-B(Traficet-EN), AJM300, Stelara (ustekinumab), Semapimod (CNI-1493)tasocitinib (CP-690550), LMW Heparin MMX, Budesonide MMX, Simponi(golimumab), MultiStem®, Gardasil HPV vaccine, Epaxal Berna (virosomalhepatitis A vaccine), surgery, such as bowel resection, strictureplastyor a temporary or permanent colostomy or ileostomy; antifungal drugssuch as nystatin (a broad spectrum gut antifungal) and eitheritraconazole (Sporanox) or fluconazole (Diflucan); alternative medicine,prebiotics and probiotics, cannabis, Helminthic therapy or ova of theTrichuris suis helminth.

Thus, treatment of psoriasis, using the agents according to at leastsome embodiments of the present invention may be combined with, forexample, any known therapeutic agent or method for treating psoriasis.Non-limiting examples of such known therapeutics for treating psoriasisinclude topical agents, typically used for mild disease, phototherapyfor moderate disease, and systemic agents for severe disease.Non-limiting examples of topical agents: bath solutions andmoisturizers, mineral oil, and petroleum jelly; ointment and creamscontaining coal tar, dithranol (anthralin), corticosteroids likedesoximetasone (Topicort), Betamethasone, fluocinonide, vitamin D3analogues (for example, calcipotriol), and retinoids. Non-limitingexamples of phototherapy: sunlight; wavelengths of 311-313 nm, psoralenand ultraviolet A phototherapy (PUVA). Non-limiting examples of systemicagents: Biologics, such as interleukin antagonists, TNF-α blockersincluding antibodies such as infliximab (Remicade), adalimumab (Humira),golimumab, certolizumab pegol, and recombinant TNF-α decoy receptor,etanercept (Enbrel); drugs that target T cells, such as efalizumab(Xannelim/Raptiva), alefacept (Ameviv), dendritic cells such Efalizumab;monoclonal antibodies (MAbs) targeting cytokines, includinganti-IL-12/IL-23 (ustekinumab (brand name Stelara)) andanti-Interleukin-17; Briakinumab (ABT-874); small molecules, includingbut not limited to ISA247; Immunosuppressants, such as methotrexate,cyclosporine; vitamin A and retinoids (synthetic forms of vitamin A);and alternative therapy, such as changes in diet and lifestyle, fastingperiods, low energy diets and vegetarian diets, diets supplemented withfish oil rich in Vitamin A and Vitamin D (such as cod liver oil), Fishoils rich in the two omega-3 fatty acids eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) and contain Vitamin E. Ichthyotherapy,Hypnotherapy, cannabis.

Thus, treatment of type 1 diabetes, using the agents according to atleast some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treating type 1diabetes. Non-limiting examples of such known therapeutics for treatingtype 1 diabetes include insulin, insulin analogs, islet transplantation,stem cell therapy including PROCHYMAL®, non-insulin therapies such asil-1beta inhibitors including Anakinra (Kineret®), Abatacept (Orencia®),Diamyd, alefacept (Ameviv®), Otelixizumab, DiaPep277 (Hsp60 derivedpeptide), Alpha 1-Antitrypsin, Prednisone, azathioprine, Ciclosporin,E1-INT (an injectable islet neogenesis therapy comprising an epidermalgrowth factor analog and a gastrin analog), statins including Zocor®,Simlup®, Simcard®, Simvacor®, Sitagliptin (dipeptidyl peptidase (DPP-4)inhibitor), Anti-CD3 mAb (e.g., Teplizumab); CTLA4-Ig (abatacept), AntiIL-1Beta (Canakinumab), Anti-CD20 mAb (e.g, rituximab).

Thus, treatment of uveitis, using the agents according to at least someembodiments of the present invention may be combined with, for example,any known therapeutic agent or method for treating uveitis. Non-limitingexamples of such known therapeutics for treating uveitis includecorticosteroids, topical cycloplegics, such as atropine or homatropine,or injection of PSTTA (posterior subtenon triamcinolone acetate),antimetabolite medications, such as methotrexate, TNF-α blockers(including infliximab, adalimumab, etanercept, golimumab, certolizumabpegol).

Thus, treatment for Sjogren's syndrome, using the agents according to atleast some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treating forSjogren's syndrome. Non-limiting examples of such known therapeutics fortreating for Sjogren's syndrome include Cyclosporine, pilocarpine(Salagen) and cevimeline (Evoxac), Hydroxychloroquine (Plaquenil),cortisone (prednisone and others) and/or azathioprine (Imuran) orcyclophosphamide (Cytoxan), Dexamethasone, Thalidomide,Dehydroepiandrosterone, NGX267, Rebamipide, FID 114657, Etanercept,Raptiva, Belimumab, MabThera (rituximab); Anakinra, intravenous immuneglobulin (IVIG), Allogeneic Mesenchymal Stem Cells (AlloMSC), Automaticneuro-electrostimulation by “Saliwell Crown”.

Thus, treatment for systemic lupus erythematosus, using the agentsaccording to at least some embodiments of the present invention may becombined with, for example, any known therapeutic agent or method fortreating for systemic lupus erythematosus. Non-limiting examples of suchknown therapeutics for treating for systemic lupus erythematosus includecorticosteroids and Disease-modifying antirheumatic drugs (DMARDs),commonly anti-malarial drugs such as plaquenil and immunosuppressants(e.g. methotrexate and azathioprine) Hydroxychloroquine, cytotoxic drugs(e.g., cyclophosphamide and mycophenolate), Hydroxychloroquine (HCQ),Benlysta (belimumab), nonsteroidal anti-inflammatory drugs, Prednisone,Cellcept, Prograf, Atacicept, Lupuzor, Intravenous Immunoglobulins(IVIGs), CellCept (mycophenolate mofetil), Orencia, CTLA4-IgG4m(RG2077), rituximab, Ocrelizumab, Epratuzumab, CNTO 136, Sifalimumab(MEDI-545), A-623 (formerly AMG 623), AMG 557, Rontalizumab, paquinimod(ABR-215757), LY2127399, CEP-33457, Dehydroepiandrosterone,Levothyroxine, abetimus sodium (UP 394), Memantine, Opiates, Rapamycin,Renal transplantation, stem cell transplantation.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, according to at least some embodiments of theinvention, may be administered as the sole active ingredient or togetherwith other drugs in immunomodulating regimens or other anti-inflammatoryagents e.g. for the treatment or prevention of allo- or xenograft acuteor chronic rejection or inflammatory or autoimmune disorders, or toinduce tolerance.

For example, it may be used in combination with a calcineurin inhibitor,e.g. cyclosporin A or FK506; an immunosuppressive macrolide, e.g.rapamycine or a derivative thereof; e.g.40-O-(2-hydroxy)ethyl-rapamycin, a lymphocyte homing agent, e.g. FTY720or an analog thereof, corticosteroids; cyclophosphamide; azathioprene;methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolicacid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof;immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies toleukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD 11a/CD18, CD7, CD25,CD 27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB ortheir ligands; or other immunomodulatory compounds, e.g. CTLA4-Ig(abatacept, ORENCIA® or belatacept), CD28-Ig, B7-H4-Ig, or othercostimulatory agents, or adhesion molecule inhibitors, e.g. mAbs or lowmolecular weight inhibitors including LFA-1 antagonists, Selectinantagonists and VLA-4 antagonists.

Where the therapeutic agents and/or a pharmaceutical compositioncomprising same, as recited herein, according to at least someembodiments of the invention are administered in conjunction with otherimmunosuppressive/immunomodulatory or anti-inflammatory therapy, e.g. asherein above specified, dosages of the co-administeredimmunosuppressant, immunomodulatory or anti-inflammatory compound willof course vary depending on the type of co-drug employed, e.g. whetherit is a steroid or a cyclosporin, on the specific drug employed, on thecondition being treated and so forth.

Treatment of malignancies using the agents of the present invention maybe combined with other treatment methods known in the art, one or moreof, for example, radiation therapy, antibody therapy, chemotherapy,photodynamic therapy, surgery or in combination therapy withconventional drugs, such as immunosuppressants or cytotoxic drugs.

A therapeutic agent or pharmaceutical composition according to at leastsome embodiments of the present invention may also be administered inconjunction with other compounds or immunotherapies. For example, thecombination therapy can include a compound of the present inventioncombined with at least one other therapeutic or immune modulatory agent,or immunostimulatory strategy, including, but not limited to, tumorvaccines, adoptive T cell therapy, Treg depletion, antibodies (e.g.bevacizumab, erbitux), peptides, pepti-bodies, small molecules,chemotherapeutic agents such as cytotoxic and cytostatic agents (e.g.paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine,temozolomide, irinotecan, 5FU, carboplatin), immunological modifierssuch as interferons and interleukins, immunostimulatory antibodies,growth hormones or other cytokines, folic acid, vitamins, minerals,aromatase inhibitors, RNAi, Histone Deacetylase Inhibitors, proteasomeinhibitors, and so forth.

According to at least some embodiments of the present invention, thereis provided use of a combination of thetherapeutic agents and/or apharmaceutical composition comprising same, as recited herein, and aknown therapeutic agent effective for treating infection.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, can be administered in combination with one ormore additional therapeutic agents used for treatment of bacterialinfections, including, but not limited to, antibiotics includingAminoglycosides, Carbapenems, Cephalosporins, Macrolides, Lincosamides,Nitrofurans, penicillins, Polypeptides, Quinolones, Sulfonamides,Tetracyclines, drugs against mycobacteria including but not limited toClofazimine, Cycloserine, Cycloserine, Rifabutin, Rifapentine,Streptomycin and other antibacterial drugs such as Chloramphenicol,Fosfomycin, Metronidazole, Mupirocin, and Tinidazole.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, can be administered in combination with one ormore additional therapeutic agents used for treatment of viralinfections, including, but not limited to, antiviral drugs such asoseltamivir (brand name Tamiflu) and zanamivir (brand name Relenza)Arbidol—adamantane derivatives (Amantadine, Rimantadine)—neuraminidaseinhibitors (Oseltamivir, Laninamivir, Peramivir, Zanamivir) nucleotideanalog reverse transcriptase inhibitor including Purine analogue guanine(Aciclovir#/Valacyclovir, Ganciclovir/Valganciclovir,Penciclovir/Famciclovir) and adenine (Vidarabine), Pyrimidine analogue,uridine (Idoxuridine, Trifluridine, Edoxudine), thymine (Brivudine),cytosine (Cytarabine); Foscarnet; Nucleoside analogues/NARTIs:Entecavir, Lamivudine, Telbivudine, Clevudine; Nucleotideanalogues/NtRTIs: Adefovir, Tenofovir; Nucleic acid inhibitors such asCidofovir; InterferonInterferon alfa-2b, Peginterferon alfa-2a;Ribavirin#/Taribavirin; antiretroviral drugs including zidovudine,lamivudine, abacavir, lopinavir, ritonavir, tenofovir/emtricitabine,efavirenz each of them alone or a various combinations, gp41(Enfuvirtide), Raltegravir, protease inhibitors such as Fosamprenavir,Lopinavir and Atazanavir, Methisazone, Docosanol, Fomivirsen,Tromantadine.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, can be administered in combination with one ormore additional therapeutic agents used for treatment of fungalinfections, including, but not limited to, antifungal drugs of thePolyene antifungals, Imidazole, triazole, and thiazole antifungals,Allylamines, Echinocandins or other anti fungal drugs.

Alternatively or additionally, an upregulating method may optionally beeffected by specifically upregulating the amount (optionally expression)in the subject of at least one of the polypeptides of the presentinvention or active portions thereof.

As is mentioned hereinabove and in the Examples section which follows,the biomolecular sequences of this aspect of the present invention maybe used as valuable therapeutic tools in the treatment of diseases,disorders or conditions in which altered activity or expression of thewild-type gene product (known protein) is known to contribute todisease, disorder or condition onset or progression. For example, incase a disease is caused by overexpression of a membrane bound-receptor,a soluble variant thereof may be used as an antagonist which competeswith the receptor for binding the ligand, to thereby terminate signalingfrom the receptor.

According to at least some embodiments, immune cells, preferably Tcells, can be contacted in vivo or ex vivo with the therapeutic agentsto modulate immune responses. The T cells contacted with the therapeuticagents can be any cell which expresses the T cell receptor, includingα/β and γ/δ T cell receptors. T-cells include all cells which expressCD3, including T-cell subsets which also express CD4 and CDS. T-cellsinclude both naive and memory cells and effector cells such as CTL.T-cells also include cells such as Th1, Tc1, Th2, Tc2, Th3, Th17, Th22,Treg, and Tr1 cells. T-cells also include NKT-cells and similar uniqueclasses of the T-cell lineage.

Inhibition of Epitope Spreading

Epitope spreading refers to the ability of B and T cell immune responseto diversify both at the level of specificity, from a single determinantto many sites on an auto antigen, and at the level of V gene usage(Monneaux, F. et al., Arthritis &amp; Rheumatism, 46(6): 1430-1438(2002). Epitope spreading is not restricted to systemic autoimmunedisease. It has been described in T cell dependent organ specificdiseases such as Diabetes mellitus type 1 and multiple sclerosis inhumans, and EAE induced experimental animals with a variety of myelinproteins.

Epitope spreading involves the acquired recognition of new epitopes inthe same self molecule as well as epitopes residing in proteins that areassociated in the same macromolecular complex. Epitope spreading can beassessed by measuring delayed-type hypersensitivity (DTH) responses,methods of which are known in the art.

One embodiment provides a method for inhibiting or reducing epitopespreading in a subject by administering to the subject an effectiveamount of the therapeutic agents. In a further embodiment any one of thetherapeutic agents inhibits epitope spreading in individuals withmultiple sclerosis. Preferably, the therapeutic agents inhibit or blockmultiple points of the inflammation pathway.

Yet another embodiment provides a method for inhibiting or reducingepitope spreading in subjects with multiple sclerosis by administeringto a subject an effective amount of the therapeutic agents to inhibit orreduce differentiation of, proliferation of, activity of, and/orcytokine production and/or secretion by Th1, Th17, Th22, and/or othercells that secrete, or cause other cells to secrete, inflammatorymolecules, including, but not limited to, IL-1beta, TNF-alpha, TGF-beta,IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

Use of the Therapeutic Agents According to at Least Some Embodiments ofthe Invention as Adjuvant for Cancer Vaccination:

Immunization against tumor-associated antigens (TAAs) is a promisingapproach for cancer therapy and prevention, but it faces severalchallenges and limitations, such as tolerance mechanisms associated withself-antigens expressed by the tumor cells. Costimulatory molecules suchas B7.1 (CD80) and B7.2 (CD86) have improved the efficacy of gene-basedand cell-based vaccines in animal models and are under investigation asadjuvant in clinical trials. This adjuvant activity can be achievedeither by enhancing the costimulatory signal or by blocking inhibitorysignal that is transmitted by negative costimulators expressed by tumorcells (Neighbors et al., 2008 J Immunother.; 31(7):644-55). According toat least some embodiments of the invention, any one of LY6G6F, VSIG10,TMEM25 and/or LSR secreted or soluble form or ECD and/or variants,and/or orthologs, and/or conjugates thereof, and/or a polyclonal ormonoclonal antibody and/or antigen binding fragments and/or conjugatescontaining same, and/or alternative scaffolds, specific to any one ofLY6G6F, VSIG10, TMEM25 and/or LSR proteins, can be used as adjuvant forcancer vaccination. According to at least some embodiments, theinvention provides methods for improving immunization against TAAs,comprising administering to a patient an effective amount of any one ofLY6G6F, VSIG10, TMEM25 and/or LSR secreted or soluble form or ECD and/orvariants, and/or orthologs, and/or conjugates thereof, and/or apolyclonal or monoclonal antibody and/or antigen binding fragmentsand/or conjugates containing same, and/or alternative scaffolds,specific to any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteins.

Use of the Therapeutic Agents According to at Least Some Embodiments ofthe Invention for Adoptive Immunotherapy:

One of the cardinal features of some models of tolerance is that oncethe tolerance state has been established, it can be perpetuated to naiverecipients by the adoptive transfer of donor-specific regulatory cells.Such adoptive transfer studies have also addressed the capacity ofT-cell subpopulations and non-T cells to transfer tolerance. Suchtolerance can be induced by blocking costimulation or upon engagement ofa co-inhibitory B7 with its counter receptor. This approach, that havebeen successfully applied in animals and is evaluated in clinical trialsin humans, (Scalapino K J and Daikh D I. PLoS One. 2009; 4(6):e6031;Riley et al., Immunity. 2009; 30(5): 656-665) provides a promisingtreatment option for autoimmune disorders and transplantation. Accordingto at least some embodiments of the invention, LY6G6F, VSIG10, TMEM25and/or LSR secreted or soluble form or ECD and/or variants, and/ororthologs, and/or conjugates thereof, and/or a polyclonal or monoclonalantibody and/or antigen binding fragments and/or conjugates containingsame, and/or alternative scaffolds, specific to any one of LY6G6F,VSIG10, TMEM25 and/or LSR proteins are used for_for adoptiveimmunotherapy. Thus, according to at least some embodiments, theinvention provides methods for in vivo or ex vivo tolerance induction,comprising administering effective amount of LY6G6F, VSIG10, TMEM25and/or LSR secreted or soluble form or ECD and/or variants, and/ororthologs, and/or conjugates thereof, and/or a polyclonal or monoclonalantibody or and/or antigen binding fragments and/or conjugatescontaining same, and/or alternative scaffolds, specific to any one ofLY6G6F, VSIG10, TMEM25 and/or LSR proteins, to a patient or toleukocytes isolated from the patient, in order to induce differentiationof tolerogenic regulatory cells; followed by ex-vivo enrichment andexpansion of said cells and reinfusion of the tolerogenic regulatorycells to said patient.

Alternatively, immune responses can be enhanced in a patient by removingimmune cells from the patient, contacting immune cells in vitro with anagent that inhibits LY6G6F, VSIG10, TMEM25 and/or LSR activity, and/orwhich inhibits the interaction of LY6G6F, VSIG10, TMEM25 and/or LSR withtheir natural binding partners, and reintroducing the in vitrostimulated immune cells into the patient. In another embodiment, amethod of modulating immune responses involves isolating immune cellsfrom a patient, transfecting them with a nucleic acid molecule encodinga form of LY6G6F, VSIG10, TMEM25 and/or LSR, such that the cells expressall or a portion of the LY6G6F, VSIG10, TMEM25 and/or LSR polypeptideaccording to various embodiments of the present invention on theirsurface, and reintroducing the transfected cells into the patient. Thetransfected cells have the capacity to modulate immune responses in thepatient.

Use of the Therapeutic Agents According to at Least Some Embodiments ofthe Invention for Immunoenhancement

1. Treatment of Cancer

The therapeutic agents provided herein are generally useful in vivo andex vivo as immune response-stimulating therapeutics. In general, thedisclosed therapeutic agent compositions are useful for treating asubject having or being predisposed to any disease or disorder to whichthe subject's immune system mounts an immune response. The ability oftherapeutic agents to modulate LY6G6F, VSIG10, TMEM25 and/or LSR immunesignals enable a more robust immune response to be possible. Thetherapeutic agents according to at least some embodiments of theinvention are useful to stimulate or enhance immune responses involvingimmune cells, such as T cells.

The therapeutic agents according to at least some embodiments of theinvention are useful for stimulating or enhancing an immune response inhost for treating cancer by administering to a subject an amount of atherapeutic agent effective to stimulate T cells in the subject.

2. Use of the Therapeutic Agents in Vaccines

The therapeutic agents according to at least some embodiments of theinvention, are administered alone or in combination with any othersuitable treatment. In one embodiment the therapeutic agents can beadministered in conjunction with, or as a component of a vaccinecomposition as described above. The therapeutic agents according to atleast some embodiments of the invention can be administered prior to,concurrently with, or after the administration of a vaccine. In oneembodiment the therapeutic agents is administered at the same time asadministration of a vaccine.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination of thetherapeutic agent, according to at least some embodiments of theinvention.

Thus, the present invention features a pharmaceutical compositioncomprising a therapeutically effective amount of a therapeutic agentaccording to at least some embodiments of the present invention.

The pharmaceutical composition according to at least some embodiments ofthe present invention is further preferably used for the treatment ofcancer, wherein the cancer may be non-metastatic, invasive ormetastatic, treatment of immune related disorder and/or infectiousdisorder.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented. Hence, the mammal to be treated herein may have beendiagnosed as having the disorder or may be predisposed or susceptible tothe disorder. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal is human.

The term “therapeutically effective amount” refers to an amount of agentaccording to the present invention that is effective to treat a diseaseor disorder in a mammal.

The therapeutic agents of the present invention can be provided to thesubject alone, or as part of a pharmaceutical composition where they aremixed with a pharmaceutically acceptable carrier.

Pharmaceutical compositions according to at least some embodiments ofthe invention also can be administered in combination therapy, i.e.,combined with other agents. For example, the combination therapy caninclude an anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSRantibody or LY6G6F, VSIG10, TMEM25 and/or LSR modulating agent accordingto at least some embodiments of the present invention, such as a solublepolypeptide conjugate containing the ectodomain of the LY6G6F, VSIG10,TMEM25 and/or LSR antigen or a small molecule such as a peptide,ribozyme, aptamer, siRNA, or other drug that binds LY6G6F, VSIG10,TMEM25 and/or LSR, combined with at least one other therapeutic orimmune modulatory agent.

A composition is said to be a “pharmaceuticall acceptable carrier” ifits administration can be tolerated by a receipient patient. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion).

Such compositions include sterile water, buffered saline (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength and optionallyadditives such as detergents and solubulizing agents (e.g., Polysorbate20, Polysorbate 80), antioxidants (e.g, ascorbic acid, sodiummetabisulfite), preservatives (e.g, Thimersol, benzyl alcohol) andblulking substances (e.g., lactose, manitol). Non-aqueoes solvents orvehicles may also be used as detailed below.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions according to at least someembodiments of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. Depending on the route ofadministration, the active compound, i.e., soluble polypeptide conjugatecontaining the ectodomain of the LY6G6F, VSIG10, TMEM25 and/or LSRantigen, monoclonal or polyclonal antibodies and antigen bindingfragments and conjugates containing same, and/or alternative scaffolds,that specifically bind any one of LY6G6F, VSIG10, TMEM25 and/or LSRproteins, or bispecific molecule, may be coated in a material to protectthe compound from the action of acids and other natural conditions thatmay inactivate the compound. The pharmaceutical compounds according toat least some embodiments of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

A pharmaceutical composition according to at least some embodiments ofthe invention also may include a pharmaceutically acceptableanti-oxidant. Examples of pharmaceutically acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsaccording to at least some embodiments of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about I percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms according to at least some embodiments of theinvention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an antibodyaccording to at least some embodiments of the invention include 1 mg/kgbody weight or 3 mg/kg body weight via intravenous administration, withthe antibody being given using one of the following dosing schedules:(i) every four weeks for six dosages, then every three months; (ii)every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kgbody weight every three weeks.

For fusion proteins as described herein, optionally a similar dosageregimen is followed; alternatively, the fusion proteins may optionallybe administered in an amount between 0.0001 to 100 mg/kg weight of thepatient/day, preferably between 0.001 to 10.0 mg/kg/day, according toany suitable timing regimen. A therapeutic composition according to atleast some embodiments of the invention can be administered, forexample, three times a day, twice a day, once a day, three times weekly,twice weekly or once weekly, once every two weeks or 3, 4, 5, 6, 7 or 8weeks. Moreover, the composition can be administered over a short orlong period of time (e.g., 1 week, 1 month, 1 year, 5 years).

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 mug/ml and in some methods about 25-300.mu.g/ml.

Alternatively, therapeutic agent can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of thetherapeutic agent in the patient. In general, human antibodies show thelongest half life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. The half-life for fusion proteinsmay vary widely. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of LY6G6F, VSIG10, TMEM25 and/orLSR soluble protein or LY6G6F, VSIG10, TMEM25 and/or LSR ectodomain orfusion protein containing same, or an anti-LY6G6F, anti-VSIG10,anti-TMEM25 and/or anti-LSR antibody according to at least someembodiments of the invention preferably results in a decrease inseverity of disease symptoms, an increase in frequency and duration ofdisease symptom-free periods, an increase in lifepan, disease remission,or a prevention or reduction of impairment or disability due to thedisease affliction. For example, for the treatment of LY6G6F, VSIG10,TMEM25 and/or LSR positive tumors, e.g., melanoma, cancers of liver,renal, brain, breast, colon, lung, ovary, pancreas, prostate, stomach,multiple myeloma and hematopoietic cancer, including but not limited tolymphoma (Hodgkin's and non Hodgkin's), acute and chronic lymphoblasticleukemia and acute and chronic myeloid leukemia a “therapeuticallyeffective dosage” preferably inhibits cell growth or tumor growth by atleast about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit tumor growth can be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto inhibit, such inhibition in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject.

One of ordinary skill in the art would be able to determine atherapeutically effective amount based on such factors as the subject'ssize, the severity of the subject's symptoms, and the particularcomposition or route of administration selected.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for therapeutic agentsaccording to at least some embodiments of the invention includeintravascular delivery (e.g. injection or infusion), intravenous,intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral,enteral, rectal, pulmonary (e.g. inhalation), nasal, topical (includingtransdermal, buccal and sublingual), intravesical, intravitreal,intraperitoneal, vaginal, brain delivery (e.g. intra-cerebroventricular,intra-cerebral, and convection enhanced diffusion), CNS delivery (e.g.intrathecal, perispinal, and intra-spinal) or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal), transmucosal(e.g., sublingual administration), administration or administration viaan implant, or other parenteral routes of administration, for example byinjection or infusion, or other delivery routes and/or forms ofadministration known in the art. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion. In a specific embodiment, a protein, a therapeutic agent or apharmaceutical composition according to at least some embodiments of thepresent invention can be administered intraperitoneally orintravenously.

Alternatively, an LY6G6F, VSIG10, TMEM25 and/or LSR specific antibody orother LY6G6F, VSIG10, TMEM25 and/or LSR drug or molecule and theirconjugates and combinations thereof that modulates a LY6G6F, VSIG10,TMEM25 and/or LSR protein activity can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition according to at least some embodiments of the invention canbe administered with a needles hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicamentsthrough the skin; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the antibodies, LY6G6F, VSIG10, TMEM25 and/orLSR soluble proteins, ectodomains, and/or fusion proteins, can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds according to at least someembodiments of the invention cross the BBB (if desired), they can beformulated, for example, in liposomes. For methods of manufacturingliposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais etal. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein Areceptor (Briscoe et al. (1995) Am. J Physiol. 1233:134); p120 (Schreieret al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L.Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994)Immunomethods 4:273.

The anti-LY6G6F, anti-VSIG10, anti-TMEM25 and anti-LSR antibodies,according to at least some embodiments of the present invention, can beused as neutralizing antibodies. A Neutralizing antibody (Nabs), is anantibody that is capable of binding and neutralizing or inhibiting aspecific antigen thereby inhibiting its biological effect, for exampleby blocking the receptors on the cell or the virus, inhibiting thebinding of the virus to the host cell. NAbs will partially or completelyabrogate the biological action of an agent by either blocking animportant surface molecule needed for its activity or by interferingwith the binding of the agent to its receptor on a target cell.

In yet another embodiment, immunoconjugates of the invention can be usedto target compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxins immunosuppressants, etc.) to cells which have LY6G6F,VSIG10, TMEM25 and/or LSR cell surface receptors by linking suchcompounds to the antibody. Thus, the invention also provides methods forlocalizing ex vivo or in vivo cells expressing LY6G6F, VSIG10, TMEM25and/or LSR (e.g., with a detectable label, such as a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor). Alternatively,the immunoconjugates can be used to kill cells which have LY6G6F,VSIG10, TMEM25 and/or LSR cell surface receptors by targeting cytotoxinsor radiotoxins to LY6G6F, VSIG10, TMEM25 and/or LSR antigen.

Diagnostic Uses of LY6G6F, VSIG10, TMEM25 and/or LSR Polypeptides andCorresponding Polynucleotides

According to some embodiments, the sample taken from a subject (patient)to perform the diagnostic assay according to at least some embodimentsof the present invention is selected from the group consisting of a bodyfluid or secretion including but not limited to blood, serum, urine,plasma, prostatic fluid, seminal fluid, semen, the external secretionsof the skin, respiratory, intestinal, and genitourinary tracts, tears,cerebrospinal fluid, synovial fluid, sputum, saliva, milk, peritonealfluid, pleural fluid, cyst fluid, secretions of the breast ductal system(and/or lavage thereof), broncho alveolar lavage, lavage of thereproductive system and lavage of any other part of the body or systemin the body; samples of any organ including isolated cells or tissues,wherein the cell or tissue can be obtained from an organ selected from,but not limited to lung, colon, ovarian and/or breast tissue; stool or atissue sample, or any combination thereof. In some embodiments, the termencompasses samples of in vivo cell culture constituents. Prior to besubjected to the diagnostic assay, the sample can optionally be dilutedwith a suitable eluant.

In some embodiments, the phrase “marker” in the context of the presentinvention refers to a nucleic acid fragment, a peptide, or apolypeptide, which is differentially present in a sample taken frompatients (subjects) having one of the herein-described diseases orconditions, as compared to a comparable sample taken from subjects whodo not have one the above-described diseases or conditions.

In some embodiments, the phrase “differentially present” refers todifferences in the quantity or quality of a marker present in a sampletaken from patients having one of the herein-described diseases orconditions as compared to a comparable sample taken from patients who donot have one of the herein-described diseases or conditions. Forexample, a nucleic acid fragment may optionally be differentiallypresent between the two samples if the amount of the nucleic acidfragment in one sample is significantly different from the amount of thenucleic acid fragment in the other sample, for example as measured byhybridization and/or NAT-based assays. A polypeptide is differentiallypresent between the two samples if the amount of the polypeptide in onesample is significantly different from the amount of the polypeptide inthe other sample. It should be noted that if the marker is detectable inone sample and not detectable in the other, then such a marker can beconsidered to be differentially present. Optionally, a relatively lowamount of up-regulation may serve as the marker, as described herein.One of ordinary skill in the art could easily determine such relativelevels of the markers; further guidance is provided in the descriptionof each individual marker below.

In some embodiments, the phrase “diagnostic” means identifying thepresence or nature of a pathologic condition. Diagnostic methods differin their sensitivity and specificity. The “sensitivity” of a diagnosticassay is the percentage of diseased individuals who test positive(percent of “true positives”). Diseased individuals not detected by theassay are “false negatives.” Subjects who are not diseased and who testnegative in the assay are termed “true negatives.” The “specificity” ofa diagnostic assay is 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

As used herein the term “diagnosis” refers to the process of identifyinga medical condition or disease by its signs, symptoms, and in particularfrom the results of various diagnostic procedures, including e.g.detecting the expression of the nucleic acids or polypeptides accordingto at least some embodiments of the invention in a biological sample(e.g. in cells, tissue or serum, as defined below) obtained from anindividual. Furthermore, as used herein the term “diagnosis” encompassesscreening for a disease, detecting a presence or a severity of adisease, providing prognosis of a disease, monitoring diseaseprogression or relapse, as well as assessment of treatment efficacyand/or relapse of a disease, disorder or condition, as well as selectinga therapy and/or a treatment for a disease, optimization of a giventherapy for a disease, monitoring the treatment of a disease, and/orpredicting the suitability of a therapy for specific patients orsubpopulations or determining the appropriate dosing of a therapeuticproduct in patients or subpopulations. The diagnostic procedure can beperformed in vivo or in vitro.

In some embodiments, the phrase “qualitative” when in reference todifferences in expression levels of a polynucleotide or polypeptide asdescribed herein, refers to the presence versus absence of expression,or in some embodiments, the temporal regulation of expression, or insome embodiments, the timing of expression, or in some embodiments, anypost-translational modifications to the expressed molecule, and others,as will be appreciated by one skilled in the art. In some embodiments,the phrase “quantitative” when in reference to differences in expressionlevels of a polynucleotide or polypeptide as described herein, refers toabsolute differences in quantity of expression, as determined by anymeans, known in the art, or in other embodiments, relative differences,which may be statistically significant, or in some embodiments, whenviewed as a whole or over a prolonged period of time, etc., indicate atrend in terms of differences in expression.

In some embodiments, the term “diagnosing” refers to classifying adisease or a symptom, determining a severity of the disease, monitoringdisease progression, forecasting an outcome of a disease and/orprospects of recovery. The term “detecting” may also optionallyencompass any of the above.

Diagnosis of a disease according to the present invention can, in someembodiments, be affected by determining a level of a polynucleotide or apolypeptide of the present invention in a biological sample obtainedfrom the subject, wherein the level determined can be correlated withpredisposition to, or presence or absence of the disease. It should benoted that a “biological sample obtained from the subject” may alsooptionally comprise a sample that has not been physically removed fromthe subject, as described in greater detail below.

In some embodiments, the term “level” refers to expression levels of RNAand/or protein or to DNA copy number of a marker of the presentinvention.

Typically the level of the marker in a biological sample obtained fromthe subject is different (i.e., increased or decreased) from the levelof the same marker in a similar sample obtained from a healthyindividual (examples of biological samples are described herein).

Numerous well known tissue or fluid collection methods can be utilizedto collect the biological sample from the subject in order to determinethe level of DNA, RNA and/or polypeptide of the marker of interest inthe subject.

Examples include, but are not limited to, fine needle biopsy, needlebiopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), andlavage. Regardless of the procedure employed, once a biopsy/sample isobtained the level of the marker can be determined and a diagnosis canthus be made.

Determining the level of the same marker in normal tissues of the sameorigin is preferably effected along-side to detect an elevatedexpression and/or amplification and/or a decreased expression, of themarker as opposed to the normal tissues.

In some embodiments, the term “test amount” of a marker refers to anamount of a marker in a subject's sample that is consistent with adiagnosis of a particular disease or condition. A test amount can beeither in absolute amount (e.g., microgram/ml) or a relative amount(e.g., relative intensity of signals).

In some embodiments, the term “control amount” of a marker can be anyamount or a range of amounts to be compared against a test amount of amarker. For example, a control amount of a marker can be the amount of amarker in a patient with a particular disease or condition or a personwithout such a disease or condition. A control amount can be either inabsolute amount (e.g., microgram/ml) or a relative amount (e.g.,relative intensity of signals).

In some embodiments, the term “detect” refers to identifying thepresence, absence or amount of the object to be detected.

In some embodiments, the term “label” includes any moiety or itemdetectable by spectroscopic, photo chemical, biochemical,immunochemical, or chemical means. For example, useful labels include32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., ascommonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens andproteins for which antisera or monoclonal antibodies are available, ornucleic acid molecules with a sequence complementary to a target. Thelabel often generates a measurable signal, such as a radioactive,chromogenic, or fluorescent signal, that can be used to quantify theamount of bound label in a sample. The label can be incorporated in orattached to a primer or probe either covalently, or through ionic, vander Waals or hydrogen bonds, e.g., incorporation of radioactivenucleotides, or biotinylated nucleotides that are recognized bystreptavadin. The label may be directly or indirectly detectable.Indirect detection can involve the binding of a second label to thefirst label, directly or indirectly. For example, the label can be theligand of a binding partner, such as biotin, which is a binding partnerfor streptavadin, or a nucleotide sequence, which is the binding partnerfor a complementary sequence, to which it can specifically hybridize.The binding partner may itself be directly detectable, for example, anantibody may be itself labeled with a fluorescent molecule. The bindingpartner also may be indirectly detectable, for example, a nucleic acidhaving a complementary nucleotide sequence can be a part of a branchedDNA molecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A.Klausner, Bio/Technology 6:1165 (1988)). Quantitation of the signal isachieved by, e.g., scintillation counting, densitometry, or flowcytometry.

Exemplary detectable labels, optionally and preferably for use withimmunoassays, include but are not limited to magnetic beads, fluorescentdyes, radiolabels, enzymes (e.g., horse radish peroxide, alkalinephosphatase and others commonly used in an ELISA), and calorimetriclabels such as colloidal gold or colored glass or plastic beads.Alternatively, the marker in the sample can be detected using anindirect assay, wherein, for example, a second, labeled antibody is usedto detect bound marker-specific antibody, and/or in a competition orinhibition assay wherein, for example, a monoclonal antibody which bindsto a distinct epitope of the marker are incubated simultaneously withthe mixture.

“Immunoassay” is an assay that uses an antibody to specifically bind anantigen. The immunoassay is characterized by the use of specific bindingproperties of a particular antibody to isolate, target, and/or quantifythe antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” or “specificallyinteracts or binds” when referring to a protein or peptide (or otherepitope), refers, in some embodiments, to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times greater than the background (non-specificsignal) and do not substantially bind in a significant amount to otherproteins present in the sample. Specific binding to an antibody undersuch conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to seminal basic protein from specific species such as rat,mouse, or human can be selected to obtain only those polyclonalantibodies that are specifically immunoreactive with seminal basicprotein and not with other proteins, except for polymorphic variants andalleles of seminal basic protein. This selection may be achieved bysubtracting out antibodies that cross-react with seminal basic proteinmolecules from other species. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

In another embodiment, this invention provides a method for detectingthe polypeptides of this invention in a biological sample, comprising:contacting a biological sample with an antibody specifically recognizinga polypeptide according to the present invention and detecting saidinteraction; wherein the presence of an interaction correlates with thepresence of a polypeptide in the biological sample.

In some embodiments of the present invention, the polypeptides describedherein are non-limiting examples of markers for diagnosing a diseaseand/or an indicative condition. Each marker of the present invention canbe used alone or in combination, for various uses, including but notlimited to, prognosis, prediction, screening, early diagnosis,determination of progression, therapy selection and treatment monitoringof a disease and/or an indicative condition.

In a related object the detected diseases will include cancers such asnon-solid and solid tumors, sarcomas and hematological malignancies.

In another related object the detected diseases will include autoimmunedisorders, rejection of any organ transplant and/or Graft versus hostdisease.

Each polypeptide/polynucleotide of the present invention can be usedalone or in combination, for various uses, including but not limited to,prognosis, prediction, screening, early diagnosis, determination ofprogression, therapy selection and treatment monitoring of diseaseand/or an indicative condition, as detailed above.

Such a combination may optionally comprise any subcombination ofmarkers, and/or a combination featuring at least one other marker, forexample a known marker. Furthermore, such a combination may optionallyand preferably be used as described above with regard to determining aratio between a quantitative or semi-quantitative measurement of anymarker described herein to any other marker described herein, and/or anyother known marker, and/or any other marker.

In some embodiments of the present invention, there are provided ofmethods, uses, devices and assays for the diagnosis of a disease orcondition. Optionally a plurality of markers may be used with thepresent invention. The plurality of markers may optionally include amarkers described herein, and/or one or more known markers. Theplurality of markers is preferably then correlated with the disease orcondition. For example, such correlating may optionally comprisedetermining the concentration of each of the plurality of markers, andindividually comparing each marker concentration to a threshold level.Optionally, if the marker concentration is above or below the thresholdlevel (depending upon the marker and/or the diagnostic test beingperformed), the marker concentration correlates with the disease orcondition. Optionally and preferably, a plurality of markerconcentrations correlates with the disease or condition.

Alternatively, such correlating may optionally comprise determining theconcentration of each of the plurality of markers, calculating a singleindex value based on the concentration of each of the plurality ofmarkers, and comparing the index value to a threshold level.

Also alternatively, such correlating may optionally comprise determininga temporal change in at least one of the markers, and wherein thetemporal change is used in the correlating step.

Also alternatively, such correlating may optionally comprise determiningwhether at least “X” number of the plurality of markers has aconcentration outside of a predetermined range and/or above or below athreshold (as described above). The value of “X” may optionally be onemarker, a plurality of markers or all of the markers; alternatively oradditionally, rather than including any marker in the count for “X”, oneor more specific markers of the plurality of markers may optionally berequired to correlate with the disease or condition (according to arange and/or threshold).

Also alternatively, such correlating may optionally comprise determiningwhether a ratio of marker concentrations for two markers is outside arange and/or above or below a threshold. Optionally, if the ratio isabove or below the threshold level and/or outside a range, the ratiocorrelates with the disease or condition.

Optionally, a combination of two or more these correlations may be usedwith a single panel and/or for correlating between a plurality ofpanels.

Optionally, the method distinguishes a disease or condition with asensitivity of at least 70% at a specificity of at least 85% whencompared to normal subjects. As used herein, sensitivity relates to thenumber of positive (diseased) samples detected out of the total numberof positive samples present; specificity relates to the number of truenegative (non-diseased) samples detected out of the total number ofnegative samples present. Preferably, the method distinguishes a diseaseor condition with a sensitivity of at least 80% at a specificity of atleast 90% when compared to normal subjects. More preferably, the methoddistinguishes a disease or condition with a sensitivity of at least 90%at a specificity of at least 90% when compared to normal subjects. Alsomore preferably, the method distinguishes a disease or condition with asensitivity of at least 70% at a specificity of at least 85% whencompared to subjects exhibiting symptoms that mimic disease or conditionsymptoms.

A marker panel may be analyzed in a number of fashions well known tothose of skill in the art. For example, each member of a panel may becompared to a “normal” value, or a value indicating a particularoutcome. A particular diagnosis/prognosis may depend upon the comparisonof each marker to this value; alternatively, if only a subset of markersis outside of a normal range, this subset may be indicative of aparticular diagnosis/prognosis. The skilled artisan will also understandthat diagnostic markers, differential diagnostic markers, prognosticmarkers, time of onset markers, disease or condition differentiatingmarkers, etc., may be combined in a single assay or device. Markers mayalso be commonly used for multiple purposes by, for example, applying adifferent threshold or a different weighting factor to the marker forthe different purposes.

In one embodiment, the panels comprise markers for the followingpurposes: diagnosis of a disease; diagnosis of disease and indication ifthe disease is in an acute phase and/or if an acute attack of thedisease has occurred; diagnosis of disease and indication if the diseaseis in a non-acute phase and/or if a non-acute attack of the disease hasoccurred; indication whether a combination of acute and non-acute phasesor attacks has occurred; diagnosis of a disease and prognosis of asubsequent adverse outcome; diagnosis of a disease and prognosis of asubsequent acute or non-acute phase or attack; disease progression (forexample for cancer, such progression may include for example occurrenceor recurrence of metastasis).

The above diagnoses may also optionally include differential diagnosisof the disease to distinguish it from other diseases, including thosediseases that may feature one or more similar or identical symptoms.

In certain embodiments, one or more diagnostic or prognostic indicatorsare correlated to a condition or disease by merely the presence orabsence of the indicators. In other embodiments, threshold levels of adiagnostic or prognostic indicators can be established, and the level ofthe indicators in a patient sample can simply be compared to thethreshold levels. The sensitivity and specificity of a diagnostic and/orprognostic test depends on more than just the analytical “quality” ofthe test—they also depend on the definition of what constitutes anabnormal result. In practice, Receiver Operating Characteristic curves,or “ROC” curves, are typically calculated by plotting the value of avariable versus its relative frequency in “normal” and “disease”populations, and/or by comparison of results from a subject before,during and/or after treatment.

According to at least some embodiments of the present invention, LY6G6F,VSIG10, TMEM25 and/or LSR protein, polynucleotide or a fragment thereof,may be featured as a biomarker for detecting disease and/or anindicative condition, as detailed above.

According to still other embodiments, the present invention optionallyand preferably encompasses any amino acid sequence or fragment thereofencoded by a nucleic acid sequence corresponding to LY6G6F, VSIG10,TMEM25 and/or LSR as described herein. Any oligopeptide or peptiderelating to such an amino acid sequence or fragment thereof mayoptionally also (additionally or alternatively) be used as a biomarker.

In still other embodiments, the present invention provides a method fordetecting a polynucleotide of this invention in a biological sample,using NAT based assays, comprising: hybridizing the isolated nucleicacid molecules or oligonucleotide fragments of at least about a minimumlength to a nucleic acid material of a biological sample and detecting ahybridization complex; wherein the presence of a hybridization complexcorrelates with the presence of the polynucleotide in the biologicalsample. Non-limiting examples of methods or assays are described below.

The present invention also relates to kits based upon such diagnosticmethods or assays. Also within the scope of the present invention arekits comprising the LY6G6F, VSIG10, TMEM25 and/or LSR protein or LY6G6F,VSIG10, TMEM25 and/or LSR conjugates or antibody compositions of theinvention (e.g., human antibodies, bispecific or multispecificmolecules, or immunoconjugates) and instructions for use. The kit canfurther contain one or more additional reagents, such as animmunosuppressive reagent, a cytotoxic agent or a radiotoxic agent, orone or more additional human antibodies according to at least someembodiments of the invention (e.g., a human antibody having acomplementary activity which binds to an epitope in the antigen distinctfrom the first human antibody).

Nucleic Acid Technology (NAT) Based Assays:

Detection of a nucleic acid of interest in a biological sample may alsooptionally be effected by NAT-based assays, which involve nucleic acidamplification technology, such as PCR for example (or variations thereofsuch as real-time PCR for example). As used herein, a “primer” definesan oligonucleotide which is capable of annealing to (hybridizing with) atarget sequence, thereby creating a double stranded region which canserve as an initiation point for DNA synthesis under suitableconditions. Amplification of a selected, or target, nucleic acidsequence may be carried out by a number of suitable methods known in theart. Non-limiting examples of amplification techniques includepolymerase chain reaction (PCR), ligase chain reaction (LCR), stranddisplacement amplification (SDA), transcription-based amplification, theq3 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci.USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202;Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al.,1989, supra). Non-limiting examples of Nucleic Acid Technology-basedassay is selected from the group consisting of a PCR, Real-Time PCR,LCR, Self-Sustained Synthetic Reaction, Q-Beta Replicase, Cycling probereaction, Branched DNA, RFLP analysis, DGGE/TGGE, Single-StrandConformation Polymorphism, Dideoxy fingerprinting, microarrays,Fluorescense In Situ Hybridization and Comparative GenomicHybridization. The terminology “amplification pair” (or “primer pair”)refers herein to a pair of oligonucleotides (oligos) of the presentinvention, which are selected to be used together in amplifying aselected nucleic acid sequence by one of a number of types ofamplification processes, preferably a polymerase chain reaction. Ascommonly known in the art, the oligos are designed to bind to acomplementary sequence under selected conditions. In one particularembodiment, amplification of a nucleic acid sample from a patient isamplified under conditions which favor the amplification of the mostabundant differentially expressed nucleic acid. In one preferredembodiment, RT-PCR is carried out on an mRNA sample from a patient underconditions which favor the amplification of the most abundant mRNA. Inanother preferred embodiment, the amplification of the differentiallyexpressed nucleic acids is carried out simultaneously. It will berealized by a person skilled in the art that such methods could beadapted for the detection of differentially expressed proteins insteadof differentially expressed nucleic acid sequences. The nucleic acid(i.e. DNA or RNA) for practicing the present invention may be obtainedaccording to well known methods.

Oligonucleotide primers of the present invention may be of any suitablelength, depending on the particular assay format and the particularneeds and targeted genomes employed. Optionally, the oligonucleotideprimers are at least 12 nucleotides in length, preferably between 15 and24 molecules, and they may be adapted to be especially suited to achosen nucleic acid amplification system. As commonly known in the art,the oligonucleotide primers can be designed by taking into considerationthe melting point of hybridization thereof with its targeted sequence(Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, 2ndEdition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols inMolecular Biology, John Wiley & Sons Inc., N.Y.).

Immunoassays

In another embodiment of the present invention, an immunoassay can beused to qualitatively or quantitatively detect and analyze markers in asample. This method comprises: providing an antibody that specificallybinds to a marker; contacting a sample with the antibody; and detectingthe presence of a complex of the antibody bound to the marker in thesample.

To prepare an antibody that specifically binds to a marker, purifiedprotein markers can be used. Antibodies that specifically bind to aprotein marker can be prepared using any suitable methods known in theart.

After the antibody is provided, a marker can be detected and/orquantified using any of a number of well recognized immunologicalbinding assays. Useful assays include, for example, an enzyme immuneassay (EIA) such as enzyme-linked immunosorbent assay (ELISA), aradioimmune assay (RIA), a Western blot assay, or a slot blot assay see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).Generally, a sample obtained from a subject can be contacted with theantibody that specifically binds the marker.

Optionally, the antibody can be fixed to a solid support to facilitatewashing and subsequent isolation of the complex, prior to contacting theantibody with a sample. Examples of solid supports include but are notlimited to glass or plastic in the form of, e.g., a microtiter plate, astick, a bead, or a microbead. Antibodies can also be attached to asolid support.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed can be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.Alternatively, the marker in the sample can be detected using anindirect assay, wherein, for example, a second, labeled antibody is usedto detect bound marker-specific antibody, and/or in a competition orinhibition assay wherein, for example, a monoclonal antibody which bindsto a distinct epitope of the marker are incubated simultaneously withthe mixture.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,marker, volume of solution, concentrations and the like. Usually theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

The immunoassay can be used to determine a test amount of a marker in asample from a subject. First, a test amount of a marker in a sample canbe detected using the immunoassay methods described above. If a markeris present in the sample, it will form an antibody-marker complex withan antibody that specifically binds the marker under suitable incubationconditions described above. The amount of an antibody-marker complex canoptionally be determined by comparing to a standard. As noted above, thetest amount of marker need not be measured in absolute units, as long asthe unit of measurement can be compared to a control amount and/orsignal.

Radio-immunoassay (RIA): In one version, this method involvesprecipitation of the desired substrate and in the methods detailedherein below, with a specific antibody and radiolabeled antibody bindingprotein (e.g., protein A labeled with 1125) immobilized on aprecipitable carrier such as agarose beads. The number of counts in theprecipitated pellet is proportional to the amount of substrate.

In an alternate version of the RIA, a labeled substrate and anunlabelled antibody binding protein are employed. A sample containing anunknown amount of substrate is added in varying amounts. The decrease inprecipitated counts from the labeled substrate is proportional to theamount of substrate in the added sample.

Enzyme linked immunosorbent assay (ELISA): This method involves fixationof a sample (e.g., fixed cells or a proteinaceous solution) containing aprotein substrate to a surface such as a well of a microtiter plate. Asubstrate specific antibody coupled to an enzyme is applied and allowedto bind to the substrate. Presence of the antibody is then detected andquantitated by a colorimetric reaction employing the enzyme coupled tothe antibody. Enzymes commonly employed in this method includehorseradish peroxidase and alkaline phosphatase. If well calibrated andwithin the linear range of response, the amount of substrate present inthe sample is proportional to the amount of color produced. A substratestandard is generally employed to improve quantitative accuracy.

Western blot: This method involves separation of a substrate from otherprotein by means of an acrylamide gel followed by transfer of thesubstrate to a membrane (e.g., nylon or PVDF). Presence of the substrateis then detected by antibodies specific to the substrate, which are inturn detected by antibody binding reagents. Antibody binding reagentsmay be, for example, protein A, or other antibodies. Antibody bindingreagents may be radiolabeled or enzyme linked as described hereinabove.Detection may be by autoradiography, colorimetric reaction orchemiluminescence. This method allows both quantitation of an amount ofsubstrate and determination of its identity by a relative position onthe membrane which is indicative of a migration distance in theacrylamide gel during electrophoresis.

Immunohistochemical analysis: This method involves detection of asubstrate in situ in fixed cells by substrate specific antibodies. Thesubstrate specific antibodies may be enzyme linked or linked tofluorophores. Detection is by microscopy and subjective evaluation. Ifenzyme linked antibodies are employed, a colorimetric reaction may berequired.

Fluorescence activated cell sorting (FACS): This method involvesdetection of a substrate in situ in cells by substrate specificantibodies. The substrate specific antibodies are linked tofluorophores. Detection is by means of a cell sorting machine whichreads the wavelength of light emitted from each cell as it passesthrough a light beam. This method may employ two or more antibodiessimultaneously.

Radio-Imaging Methods

These methods include but are not limited to, positron emissiontomography (PET) single photon emission computed tomography (SPECT).Both of these techniques are non-invasive, and can be used to detectand/or measure a wide variety of tissue events and/or functions, such asdetecting cancerous cells for example. Unlike PET, SPECT can optionallybe used with two labels simultaneously. SPECT has some other advantagesas well, for example with regard to cost and the types of labels thatcan be used. For example, U.S. Pat. No. 6,696,686 describes the use ofSPECT for detection of breast cancer, and is hereby incorporated byreference as if fully set forth herein.

Theranostics:

The term theranostics describes the use of diagnostic testing todiagnose the disease, choose the correct treatment regime according tothe results of diagnostic testing and/or monitor the patient response totherapy according to the results of diagnostic testing. Theranostictests can be used to select patients for treatments that areparticularly likely to benefit them and unlikely to produceside-effects. They can also provide an early and objective indication oftreatment efficacy in individual patients, so that (if necessary) thetreatment can be altered with a minimum of delay. For example: DAKO andGenentech together created HercepTest and Herceptin (trastuzumab) forthe treatment of breast cancer, the first theranostic test approvedsimultaneously with a new therapeutic drug. In addition to HercepTest(which is an immunohistochemical test), other theranostic tests are indevelopment which use traditional clinical chemistry, immunoassay,cell-based technologies and nucleic acid tests. PPGx's recently launchedTPMT (thiopurine S-methyltransferase) test, which is enabling doctors toidentify patients at risk for potentially fatal adverse reactions to6-mercaptopurine, an agent used in the treatment of leukemia. Also, NovaMolecular pioneered SNP genotyping of the apolipoprotein E gene topredict Alzheimer's disease patients' responses to cholinomimetictherapies and it is now widely used in clinical trials of new drugs forthis indication. Thus, the field of theranostics represents theintersection of diagnostic testing information that predicts theresponse of a patient to a treatment with the selection of theappropriate treatment for that particular patient.

Surrogate Markers:

A surrogate marker is a marker, that is detectable in a laboratoryand/or according to a physical sign or symptom on the patient, and thatis used in therapeutic trials as a substitute for a clinicallymeaningful endpoint. The surrogate marker is a direct measure of how apatient feels, functions, or survives which is expected to predict theeffect of the therapy. The need for surrogate markers mainly arises whensuch markers can be measured earlier, more conveniently, or morefrequently than the endpoints of interest in terms of the effect of atreatment on a patient, which are referred to as the clinical endpoints.Ideally, a surrogate marker should be biologically plausible, predictiveof disease progression and measurable by standardized assays (includingbut not limited to traditional clinical chemistry, immunoassay,cell-based technologies, nucleic acid tests and imaging modalities).

The therapeutic compositions (e.g., human antibodies, multispecific andbispecific molecules and immunoconjugates) according to at least someembodiments of the invention which have complement binding sites, suchas portions from IgG1, -2, or -3 or IgM which bind complement, can alsobe used in the presence of complement. In one embodiment, ex vivotreatment of a population of cells comprising target cells with abinding agent according to at least some embodiments of the inventionand appropriate effector cells can be supplemented by the addition ofcomplement or serum containing complement. Phagocytosis of target cellscoated with a binding agent according to at least some embodiments ofthe invention can be improved by binding of complement proteins. Inanother embodiment target cells coated with the compositions (e.g.,human antibodies, multispecific and bispecific molecules) according toat least some embodiments of the invention can also be lysed bycomplement. In yet another embodiment, the compositions according to atleast some embodiments of the invention do not activate complement.

The therapeutic compositions (e.g., human antibodies, multispecific andbispecific molecules and immunoconjugates) according to at least someembodiments of the invention can also be administered together withcomplement. Thus, according to at least some embodiments of theinvention there are compositions, comprising human antibodies,multispecific or bispecific molecules and serum or complement. Thesecompositions are advantageous in that the complement is located in closeproximity to the human antibodies, multispecific or bispecificmolecules. Alternatively, the human antibodies, multispecific orbispecific molecules according to at least some embodiments of theinvention and the complement or serum can be administered separately.

The present invention is further illustrated by the following examples.This information and examples is illustrative and should not beconstrued as further limiting. The contents of all figures and allreferences, patents and published patent applications cited throughoutthis application are expressly incorporated herein by reference.

EXAMPLES Example 1

Expression Pattern of the Proteins According to at Least SomeEmbodiments of the Invention Using MED Discovery Engine:

MED is a proprietary software platform for collection of publicgene-expression data, normalization, annotation and performance ofvarious queries. Expression data from the most widely used Affymetrixmicroarrays is downloaded from the Gene Expression Omnibus(GEO—www.ncbi.nlm.nih.gov/GEO). Data is multiplicatively normalized bysetting the 95 percentile to a constant value (normalizedexpression=1200), and noise is filtered by setting the lower 30% to 0.Experiments are annotated, first automatically, and then manually, toidentify tissue and condition, and chips are grouped according to thisannotation, and cross verification of this grouping by comparing theoverall expression pattern of the genes of each chip to the overallaverage expression pattern of the genes in this group. Each probeset ineach group is assigned an expression value which is the median of theexpressions of that probeset in all chips included in the group. Thevector of expression of all probesets within a certain group forms thevirtual chip of that group, and the collection of all such virtual chipsis a virtual panel. The panel (or sub-panels) can be queried to identifyprobesets with a required behavior (e.g. specific expression in asub-set of tissues, or differential expression between disease andhealthy tissues). These probesets are linked to LEADS contigs and toRefSeqs (http://www.ncbi.nlm.nih.gov/RefSeq/) by probe-level mapping,for further analysis.

The Affymetrix platforms that are downloaded are HG-U95A and the HG-U133family (A,B, A2.0 and PLUS 2.0). Three virtual panels were created: U95and U133 Plus 2.0, based on the corresponding Affymetrix platforms, andU133 which uses the set of common probesets for HG-U133A, HG-U133A2.0and HG-U133 PLUS 2.0+.

The results of the MED discovery engine are presented in scatter plots.The scatter plot is a compact representation of a given panel(collection of groups). The y-axis is the (normalized) expression andthe x-axis describes the groups in the panel. For each group, the medianexpression is represented by a solid marker, and the expression valuesof the different chips in the group are represented by small dashes(“-”). The groups are ordered and marked as follows—“Other” groups (e.g.benign, non-cancer diseases, etc.) with a triangle, Treated cells with asquare, Normal with a circle, Matched with a cross, and Cancer with adiamond. The number of chips in each group is also written adjacent toits name.

The MED discovery engine was used to assess the expression of VSIG10transcripts. Expression data for Affymetrix probe sets 220137_atrepresenting the VSIG10 gene data is shown in FIG. 3 (for all figuresrelated to the MED discovery engine, a division was made into “A”, B″,etc for reasons of space only, so as to be able to show all proberesults). As evident from the scatter plot, presented in FIG. 3, theexpression of VSIG10 transcripts detectable with the above probe setswas observed in several groups of cells from the immune system, mainlyin leukocytes. In various cancer conditions, differential expression wasobserved, for example on CD10+ leukocytes from ALL (Acute LymphoblasticLeukemia) and BM-CD34+cells from AML (Acute Myeloid Leukemia) cells.

FIG. 3 shows a scatter plot, demonstrating the expression of VSIG10transcripts that encode the VSIG10 proteins, on a virtual panel of alltissues and conditions using MED discovery engine.

MED discovery engine was used to assess the expression of LSRtranscripts. Expression data for Affymetrix probe sets 208190_s_atrepresenting the LSR gene data is shown in FIG. 4. As evident from thescatter plot, presented in FIG. 4, the expression of LSR transcriptsdetectable with the above probe sets was observed in several groups ofcells from the immune system, mainly in bone marrow cells. Highexpression of LSR transcripts was also observed in various cancerousconditions of tissues, such as in breast, lung, ovary, pancreas,prostate and skin cancers.

FIG. 4 shows a scatter plot, demonstrating the expression of LSRtranscripts that encode the LSR proteins, on a virtual panel of alltissues and conditions using MED discovery engine.

Example 2 Methods Used to Analyze the Expression of the RNA EncodingLY6G6F, VSIG10, TMEM25 and/or LSR Proteins

The targets according to at least some embodiments of the presentinvention were tested with regard to their expression in variouscancerous and non-cancerous tissue samples. A description of the samplesused in the Ovary cancer testing panel is provided in Table 1 below. Adescription of the samples used in the Breast cancer testing panel isprovided in Table 2 below. A description of the samples used in the Lungcancer testing panel is provided in Table 3. A description of thesamples used in the Healthy testing panel is provided in Table 4. Adescription of the samples used in the Kidney cancer testing panel isprovided in Table 5. A description of the samples used in the Livercancer testing panel is provided in Table 6. Tests were then performedas described in the Materials and Methods section below.

Materials and Methods

RNA Preparation—

RNA was obtained from ABS (Wilmington, Del. 19801, USA,http://www.absbioreagents.com), BioChain Inst. Inc. (Hayward, Calif.94545 USA, www.biochain.com), GOG for ovary samples-Pediatic CooperativeHuman Tissue Network, Gynecologic Oncology Group Tissue Bank, ChildrenHospital of Columbus (Columbus Ohio 43205 USA), Ambion (Austin, Tex.78744 USA, http://www.ambion.com), Analytical Biological Services Inc.(Wilmington, Del. 19801 USA, www.absbioreagents.com), Asternad (Detroit,Mich. 48202-3420, USA, www.asterand.com), Genomics Collaborative Inc. aDivision of Seracare (Cambridge, Mass. 02139, USA, www.genomicsinc.com),The Tel Aviv Sourasky Medical Center Ichilov Hospital (Tel-Aviv, ISRAEL,www.tasmc.org.il/e/) and from The Chaim Sheba Medical Center(Tel-Hashomer, ISRAEL, eng.sheba.co.il). RNA samples were obtained frompatients or from postmortem. All total RNA samples were treated withDNasel (Ambion).

RT-PCR for Ovary, Kidney and Healthy Panel—

10 ug of Purified RNA was mixed with Random Hexamer primers (AppliedBiosystems, according to manufactures instructions), 4 mM dNTPs, 12.5 μlof 10× MultiScribe™ buffer (Applied Biosystems), 6 μl (50U/μL) RNasin(Promega) and 6 μl (50U/μL) of MultiScribe (Applied Biosystems) in atotal volume of 125 μl. The reaction was incubated for 10 mM at 25° C.,followed by further incubation at 37° C. for 2 hours. Then, the mixturewas inactivated at 85° C. for 5 sec. The resulting cDNA was diluted1:10-1:40 (depend on the panel calibration) in TE buffer (10 mM TrispH=8, 1 mM EDTA pH=8).

Real-Time RT-PCR analysis was carried out as described below-cDNA (5μl), prepared as described above, was used as a template for Real-TimePCR reactions (final volume of 20 μl) using the SYBR Green I assay (PEApplied Biosystem) with specific primers and UNG Enzyme (Eurogentech orABI or Roche). The amplification was effected as follows: 50° C. for 2min, 95° C. for 10 mM, and then 40 cycles of 95° C. for 15 sec, followedby 60° C. for 30 sec, following by dissociation step. Detection wasperformed using the PE Applied Biosystem SDS 7000. The cycle in whichthe reactions achieved a threshold level of fluorescence (Ct=ThresholdCycle, described in detail below) was registered and was used tocalculate the relative transcript quantity in the RT reactions. Therelative quantity was calculated using the equation Q=efficiencŷ−Ct. Theefficiency of the PCR reaction was calculated from a standard curve,created by using different dilutions of several reverse transcription(RT) reactions. To minimize inherent differences in the RT reaction, theresulting relative quantities were normalized using a normalizationfactor calculated in the following way:

The expression of several housekeeping (HSKP) genes was checked in everypanel. The relative quantity (Q) of each housekeeping gene in eachsample, calculated as described above, was divided by the medianquantity of this gene in all panel samples to obtain the “Relative Q relto MED”. Then, for each sample the median of the “relative Q rel to MED”of the selected housekeeping genes was calculated and served asnormalization factor of this sample for further calculations.

For each RT sample, the expression of the specific amplicon wasnormalized to the normalization factor calculated from the expression ofdifferent housekeeping genes. Housekeeping genes (HSKG) used for Ovary,Kidney, Lung, Liver, Breast and Healthy panels are listed in Table 7.

The HSKGs that were used for Ovary and Healthy panels calibration are:HPRT1, SDHA and G6PD; The HSKP genes used for Kidney and Liver panelcalibration are: G6PD, PBGD and SDHA; The HSKP genes used for Lung panelcalibration are: UBC, PBGD, HPRT and SDHA; The HSKP genes used forBreast panel calibration are: G6PD, PBGD, RPL19 and SDHA;

TABLE 1 Ovary RNA details: sample name Source sample_id DIAGNOSISCANCER_STAGE 1-As-SI-SER Asterand 23074 SEROUS STAGE I ADENOCARCINOMA2-As-SI-SER Asterand 22653 SEROUS STAGE I ADENOCARCINOMA 3-As-SIB-SERAsterand 18700 SEROUS STAGE IB ADENOCARCINOMA 4-As-SIB-SER Asterand17646 SEROUS STAGE IB ADENOCARCINOMA 5-As-SIC-SER Asterand 15644 SEROUSSTAGE IC ADENOCARCINOMA 6-GC-SIIB-SER GCI-1st_del 7B3DP SEROUS STAGE IIBADENOCARCINOMA 7-As-SIIIC-SER Asterand 13268 SEROUS STAGE IIICADENOCARCINOMA 8-GC-SIIIC-SER GCI-1st_del 3NTIS SEROUS STAGE IIICADENOCARCINOMA 9-GC-SIIIC-SER GCI-1st_del CEJUS SEROUS STAGE IIICADENOCARCINOMA 10-GC-SIIIC-SER GCI-1st_del 1HI5H SEROUS STAGE IIICADENOCARCINOMA 11-GC-SIIIC-SER GCI-1st_del 7RMHZ SEROUS STAGE IIICADENOCARCINOMA 12-GC-SIIIC-SER GCI-1st_del 4WAAB SEROUS STAGE IIICADENOCARCINOMA 13-GC-SIIIC-SER GCI-1st_del 79Z67 SEROUS STAGE IIICADENOCARCINOMA 14-GC-SIIIC-SER GCI-1st_del DDSNL SEROUS STAGE IIICADENOCARCINOMA 15-GC-SIV-SER GCI-1st_del DH8PH SEROUS STAGE IVADENOCARCINOMA 16-GC-SIA-ENDO GCI-1st_del E2WKF ENDOMETROID STAGE IAADENOCARCINOMA 17-GC-SIA-ENDO GCI-1st_del HZ2EY ENDOMETROID STAGE IAADENOCARCINOMA 18-GC-SIA-ENDO GCI-1st_del RWOIV ENDOMETROID STAGE IAADENOCARCINOMA 19-GC-SIIA-ENDO GCI-1st_del 1U52X ENDOMETROID STAGE IIAADENOCARCINOMA 20-GC-SIIB-ENDO GCI-1st_del A17WS ENDOMETROID STAGE IIBADENOCARCINOMA 21-GC-SIIIC-ENDO GCI-1st_del 1VT3I ENDOMETROID STAGE IIICADENOCARCINOMA 22-GC-SIIIC-ENDO GCI-1st_del PZQXH ENDOMETROID STAGE IIICADENOCARCINOMA 23-GC-SIV-ENDO GCI-1st_del I8VHZ ENDOMETROID STAGE IVADENOCARCINOMA 24-GC-SIC-MUC GCI-1st_del IMDA1 MUCINOUS STAGE ICADENOCARCINOMA 25-As-SIC-MUC Asterand 12742 MUCINOUS STAGE ICADENOCARCINOMA 26-AB-SIC-MUC ABS A0139 Mucinous Stage ICcystadenocarcinoma 27- AB-SIIIA-MUC ABS USA-00273 Papillary mucinousSTAGE IIIA cystadenocarcinoma 28-GC-SIIIA-MUC GCI-2nd_del RAFCW MUCINOUSSTAGE IIIA ADENOCARCINOMA 29-As-SIIIC-MUC Asterand 23177 MUCINOUS STAGEIIIC ADENOCARCINOMA 30-As-SIIIC-MUC Asterand 16103 MUCINOUS STAGE IIICADENOCARCINOMA 31-GC-SIA-BRD GCI-3rd_del SC656 MUCINOUS STAGE IABORDERLINE TUMOR 32-GC-SIA-BRD GCI-3rd_del 3D5FO MUCINOUS STAGE IABORDERLINE TUMOR 33-GC-SIA-BRD GCI-3rd_del 7JP3F MUCINOUS STAGE IABORDERLINE TUMOR 34-GC-Muc-BNG GCI-1st_del QLIKY BENIGN MUCINOUSCYSTADENOMA 35-As-Muc-BNG Asterand 16870 BENIGN MUCINOUS CYSTADENOMA36-GC-Muc-BNG GCI-1st_del 943EC BENIGN MUCINOUS CYSTADENOMA37-GC-Muc-BNG GCI-2nd_del JO8W7 BENIGN MUCINOUS CYSTADENOMA38-As-Ser-BNG Asterand 17016 BENIGN SEROUS IA CYSTADENOMA 39-GO-Ser-BNGGOG 99-06-G039 BENIGN SEROUS CYSTADENOMA 40-GC-Ser-BNG GCI-2nd_del DQQ2FBENIGN SEROUS CYSTADENOFIBROMA 41-As-BM-N Asterand 15690 NORMAL OVARY-BM42-As-BM-N Asterand 16850 NORMAL OVARY-BM 43-As-BM-N Asterand 16848NORMAL OVARY-BM 44-GC-PS-N GCI-4th_del WPU1U NORMAL OVARY-PS 45-GC-PS-NGCI-4th_del Y9VHI NORMAL OVARY-PS 46-GC-PS-N GCI-4th_del 76VM9 NORMALOVARY-PS 47-GC-PS-N GCI-1st_del DWHTZ NORMAL OVARY-PS 48-GC-PS-NGCI-1st_del SJ2R2 NORMAL OVARY-PS 49-GC-PS-N GCI-4th_del 9RQMN NORMALOVARY-PS 50-GC-PS-N GCI-1st_del TOAE5 NORMAL OVARY-PS 51-GC-PS-NGCI-1st_del TW9PM NORMAL OVARY-PS 52-GC-PS-N GCI-4th_del 2VND2 NORMALOVARY-PS 53-GC-PS-N GCI-1st_del L629F NORMAL OVARY-PS 54-GC-PS-NGCI-1st_del XLB23 NORMAL OVARY-PS 55-GC-PS-N GCI-1st_del IDUVY NORMALOVARY-PS 56-GC-PS-N GCI-4th_del ZCXAD NORMAL OVARY-PS 57-GC-PS-NGCI-4th_del PEQ6C NORMAL OVARY-PS 58-GC-PS-N GCI-1st_del DD73B NORMALOVARY-PS 59-GC-PS-N GCI-4th_del E2UF7 NORMAL OVARY-PS 60-GC-PS-NGCI-4th_del 4YG5P NORMAL OVARY-PS 61-GC-PS-N GCI-1st_del FDPL9 NORMALOVARY-PS 62-Bc-PM-N BioChain A503274 NORMAL OVARY-PM 63-Bc-PM-N BioChainA504086 NORMAL OVARY-PM 64-Ic-PM-N Ichilov CG-188-7 NORMAL OVARY-PM65-GO-SIIIC-SER GOG 2001-12-G035 Serous adenocarcinoma Stage 3C66-AB-SIIIC-SER ABS N0021 Papillary serous Stage 3C adenocarcinoma67-BC-SER BioChain A503175 Serous papillary cystadenocarcinoma 68-Bc-SERBiochain A406023 Adenocarcinoma 69-Bc-SER Biochain A407068Adenocarcinoma 70-AB-SER ABS ILS-7286 Papillary UN cystadenocarcinoma71-AB-SER ABS A0106 adenocarcinoma UN 72-AB-SER ABS ILS-1431 Papillaryadenocarcinoma UN 73-Bc-SER BioChain A503176 Serous papillarycystadenocarcinoma 74-AB-SER ABS ILS-1408 Papillary adenocarcinoma UN75-Bc-SER Biochain A407069 Adenocarcinoma 76-AB-SER ABS ILS-1406Papillary adenocarcinoma UN 77-GO-Ser Mix GOG 2002-05-G509 Mixed serousand Stage3C SIIIC-OTR endometrioid adenocarcinoma of mullerian 78-Bc-MUCBioChain A504083 Mucinous adenocarcinoma 79-Bc-MUC BioChain A504084Mucinous adenocarcinoma 80-Bc-Car-OTR BioChain A407065 Carcinoma81-GO-Clear cell GOG 2001-10-G002 Clear cell Stage 3A SIIIA-OTRadenocarcinoma 82-AB-BRD ABS VNM-00187 Mucinous cystadenocarcinoma withlow malignant 83-GO-SIA-BRD GOG 98-08-G001 Endometroid Stage 1Aadenocarcinoma of borderline malignancy

TABLE 2 Breast RNA details: sample name Source sample_id SampleDIAGNOSIS CANCER_STAGE 1-As-DCIS S0 Asterand 19723 Ductal CarcinomaSTAGE 0 In Situ(DCIS) 2-GC-IDC SI GCI-1st_del 5IRTK INFILTRATING STAGE IDUCTAL CARCINOMA 3-(42)-AB-IDC SI ABS 6005020031T INFILTRATING STAGE IDUCTAL CARCINOMA 4-(7)-AB-IDC SI ABS 7263T INFILTRATING STAGE I DUCTALCARCINOMA 5-GC-IDC SI GCI-1st_del DSI52 INFILTRATING STAGE I DUCTALCARCINOMA 6-GC-IDC SI GCI-1st_del S2GBY INFILTRATING STAGE I DUCTALCARCINOMA 7-GC-IDC SI GCI-1st_del POPHP INFILTRATING STAGE I DUCTALCARCINOMA 8-GC-IDC SI GCI-1st_del I2YLE INFILTRATING STAGE I DUCTALCARCINOMA 9-As-IDC SI Asterand 17959 INFILTRATING STAGE I DUCTALCARCINOMA 10-(12)-AB-IDC SIIA ABS 1432T INFILTRATING STAGE IIA DUCTALCARCINOMA 11-As-IDC SIIA Asterand 17138 INFILTRATING STAGE IIA DUCTALCARCINOMA 12-GC-IDC SIIA GCI-1st_del YSZ67 INFILTRATING STAGE IIA DUCTALCARCINOMA 13-(6)-AB-IDC SIIA ABS 7238T INFILTRATING STAGE IIA DUCTALCARCINOMA 14-(26)-AB-IDC SIIA ABS 7249T INFILTRATING STAGE IIA DUCTALCARCINOMA 15-GC-IDC SIIA GCI-1st_del UT3SE INFILTRATING STAGE IIA DUCTALCARCINOMA 16-GC-IDC SIIA GCI-1st_del PVSYX INFILTRATING STAGE IIA DUCTALCARCINOMA 17-GC-IDC SIIA GCI-1st_del GETCV INFILTRATING STAGE IIA DUCTALCARCINOMA 18-(27)-AB-IDC SIIA ABS 4907020072T INFILTRATING STAGE IIADUCTAL CARCINOMA 19-GC-IDC SIIB GCI-1st_del SE5BK INFILTRATING STAGE IIBDUCTAL CARCINOMA 20-GC-IDC SIIB GCI-1st_del OLKL4 INFILTRATING STAGE IIBDUCTAL CARCINOMA 21-GC-IDC SIIB GCI-1st_del VK1EJ INFILTRATING STAGE IIBDUCTAL CARCINOMA 22-GC-IDC SIIB GCI-1st_del 3Z5Z4 INFILTRATING STAGE IIBDUCTAL CARCINOMA 23-(13)-AB-IDC SIIB ABS A0133T INFILTRATING STAGE IIBDUCTAL CARCINOMA 24-GC-IDC SIIB GCI-1st_del J5MPN INFILTRATING STAGE IIBDUCTAL CARCINOMA 25-GC-IDC SIIB GCI-1st_del 54NTA INFILTRATING STAGE IIBDUCTAL CARCINOMA 27-GC-IDC SIIIA GCI-1st_del RD3F9 INFILTRATING STAGEIIIA DUCTAL CARCINOMA 28-(17)-AB-IDC ABS 4904020036T INFILTRATING STAGEIIIA SIIIA DUCTAL CARCINOMA 29-(16)-AB-IDC IIIA ABS 4904020032TINFILTRATING STAGE IIIA DUCTAL CARCINOMA 30-(15)-AB-IDC ABS 7259TINFILTRATING STAGE IIIA SIIIA DUCTAL CARCINOMA 31-GC-IDC SIIIAGCI-1st_del YOLOF INFILTRATING STAGE IIIA DUCTAL CARCINOMA 32-GC-IDCSIIIB GCI-1st_del 4W2NY INFILTRATING STAGE IIIB DUCTAL CARCINOMA33-GC-IDC SIIIB GCI-1st_del YQ1WW INFILTRATING STAGE IIIB DUCTALCARCINOMA 34-GC-IDC SIIIB GCI-1st_del KIOE7 INFILTRATING STAGE IIIBDUCTAL CARCINOMA 35-As-ILC SI Asterand 17090 INFILTRATING STAGE ILOBULAR CARCINOMA 36-GC-ILC SIIA GCI-1st_del I35US INFILTRATING STAGEIIA LOBULAR CARCINOMA 37-GC-ILC SIIB GCI-1st_del IS84Y INFILTRATINGSTAGE IIB LOBULAR CARCINOMA 38-(52)-Bc-ILC Biochain A605360 INFILTRATINGLOBULAR CARCINOMA 39-As-BNG Asterand 11975 FIBROADENOMA 40-GC-BNGGCI-2nd_del ZT15M FIBROADENOMA 41-GC-BNG GCI-2nd_del NNP3Q FIBROADENOMA42-GC-BNG GCI-2nd_del QK8IY FIBROADENOMA 43-GC-N PS GCI-1st_del 83LO7NORMAL BREAST-PS 45-GC-N PS GCI-2nd_del O6JBJ NORMAL BREAST-PS 46-GC-NPS GCI-2nd_del E6UDD NORMAL BREAST-PS 47-GC-N PS GCI-1st_del DHLR1NORMAL BREAST-PS 48-GC-N PS GCI-2nd_del JHQEH NORMAL BREAST-PS49-(63)-Am-N PS Ambion 26486 NORMAL BREAST-PS 50-GC-N PS GCI-2nd_delONBFK NORMAL BREAST-PS 51-GC-N PS GCI-1st_del TG6J6 NORMAL BREAST-PS52-As-N PS Asterand 14398 NORMAL BREAST-PS 54-GC-N PS GCI-2nd_del AJGXVNORMAL BREAST-PS 56-GC-N PS GCI-1st_del HLCZX NORMAL BREAST-PS 58-GC-NPS GCI-1st_del FGV8P NORMAL BREAST-PS 59-As-N PS Asterand 9264 NORMALBREAST-PS 60-(57)-Bc-N PM Biochain A609233 NORMAL BREAST-PM 61-(59)-Bc-NPM Biochain A607155 NORMAL BREAST-PM 62-(60)-Bc-N PM Biochain A609234NORMAL BREAST-PM 63-(66)-Am-N PM Ambion 36678 NORMAL BREAST-PM64-(64)-Am-N PM Ambion 23036 NORMAL BREAST-PM 66-(67)-Am-N PM Ambion073P010602086A NORMAL BREAST-PM 67-(58)-Bc-N PM Biochain A609232 NORMALBREAST-PM 68-As-N PM Asterand 8862 NORMAL BREAST-PM 69-As-N PM Asterand8457 NORMAL BREAST-PM 70-(43)-Bc-IDC Biochain A609183 INFILTRATINGDUCTAL CARCINOMA 71-(54)-Bc-IDC Biochain A605353 INFILTRATING DUCTALCARCINOMA 72-(55)-Bc-IDC ABS A609179 INFILTRATING DUCTAL CARCINOMA73-(47)-Bc-IDC Biochain A609221 INFILTRATING DUCTAL CARCINOMA74-(48)-Bc-IDC Biochain A609222 INFILTRATING DUCTAL CARCINOMA75-(53)-Bc-IDC Biochain A605151 INFILTRATING DUCTAL CARCINOMA76-(61)-Bc-IDC Biochain A610029 INFILTRATING DUCTAL CARCINOMA77-(46)-Bc-Carci Biochain A609177 Carcinoma 78-(62)-Bc-IDC BiochainA609194 INFILTRATING DUCTAL CARCINOMA 79-(32)-AB-Muc Ambion 7116TMucinous STAGE IIA Carci SIIA carcinoma 80-(49)-Bc-IDC Biochain A609223INFILTRATING DUCTAL CARCINOMA 81-(45)-Bc-IDC Biochain A609181INFILTRATING DUCTAL CARCINOMA 82-(50)-Bc-IDC Biochain A609224INFILTRATING DUCTAL CARCINOMA 83-(44)-Bc-IDC Biochain A609198INFILTRATING DUCTAL CARCINOMA 84-(51)-Bc-IDC Biochain A605361INFILTRATING DUCTAL CARCINOMA 85-(31)-Ic-IDC Ambion CG-154 INFILTRATINGDUCTAL CARCINOMA

TABLE 3 Lung Panel RNA Details sample name Source sample_id DIAGNOSISCANCER_STAGE 1-GC-BAC-SIA GCI-1st_del 7Z9V4 ADENOCARCINOMA STAGE IA2-GC-BAC-SIB GCI-1st_del ZW2AQ ADENOCARCINOMA STAGE IB 4-GC-Adeno-SIAGCI-1st_del 3MOPL ADENOCARCINOMA STAGE IA 5-GC-Adeno-SIA GCI-1st_delKOJXD ADENOCARCINOMA STAGE IA 6-GC-Adeno-SIA GCI-1st_del X2Q44ADENOCARCINOMA STAGE IA 8-GC-Adeno-SIA GCI-1st_del BS9AF ADENOCARCINOMASTAGE IA 9-GC-Adeno-SIA GCI-1st_del UCLOA ADENOCARCINOMA STAGE IA10-GC-Adeno-SIA GCI-1st_del BVYK3 ADENOCARCINOMA STAGE IA11-GC-Adeno-SIB GCI-1st_del U4DM4 ADENOCARCINOMA STAGE IB12-GC-Adeno-SIB GCI-1st_del OWX5Y ADENOCARCINOMA STAGE IB13-GC-Adeno-SIIA GCI-1st_del XYY96 ADENOCARCINOMA STAGE IIA14-GC-Adeno-SIIA GCI-1st_del SO7B1 ADENOCARCINOMA STAGE IIA15-GC-Adeno-SIIIA GCI-1st_del QANSY ADENOCARCINOMA STAGE IIIA18-(76)-Bc-Adeno Biochain A609218 ADENOCARCINOMA 19-As-Sq-S0 Asterand9220 Squamous Cell Carcinoma Occult 20-GC-Sq-SIA GCI-1st_del U2QHSSquamous Cell Carcinoma STAGE IA 21-GC-Sq-SIB GCI-2nd_del TRQR7 SquamousCell Carcinoma STAGE IB 22-As-Sq-SIB Asterand 17581 Squamous CellCarcinoma STAGE IB 23-As-Sq-SIB Asterand 18309 Squamous Cell CarcinomaSTAGE IB 24-As-Sq-SIB Asterand 9217 Squamous Cell Carcinoma STAGE IB25-GC-Sq-SIIB GCI-1st_del RXQ1P Squamous Cell Carcinoma STAGE IIB26-GC-Sq-SIIB GCI-1st_del KB5KH Squamous Cell Carcinoma STAGE IIB27-GC-Sq-SIIIA GCI-1st_del LAYMB Squamous Cell Carcinoma STAGE IIIA30-(19)-Bc-Sq Biochain A408175 Squamous Cell Carcinoma 31-(78)-Bc-SqBiochain A607125 Squamous Cell Carcinoma 33-(80)-Bc-Sq Biochain A609163Squamous Cell Carcinoma 34-(18)-Bc-Sq Biochain A503387 Squamous CellCarcinoma 35-(81)-Bc-Sq Biochain A609076 Squamous Cell Carcinoma36-GC-LCC-SIA GCI-1st_del AF8AL LARGE CELL CARCINOMA STAGE IA37-GC-LCC-SIB GCI-1st_del O62XU LARGE CELL CARCINOMA STAGE IB38-GC-LCC-SIB GCI-2nd_del OLOIM LARGE CELL CARCINOMA STAGE IB39-GC-LCC-SIIB GCI-4th_del 1ZWSV LARGE CELL CARCINOMA STAGE IIB41-GC-LCC-SIIB GCI-1st_del 38B4D LARGE CELL CARCINOMA STAGE IIB42-GC-SCC-SIB GCI-1st_del QPJQL SMALL CELL CARCINOMA STAGE IB43-(32)-Bc-SCC Biochain A501391 SMALL CELL CARCINOMA 44-(30)-Bc-SCCBiochain A501389 SMALL CELL CARCINOMA 45-(83)-Bc-SCC Biochain A609162SMALL CELL CARCINOMA 46-(86)-Bc-SCC Biochain A608032 SMALL CELLCARCINOMA 47-(31)-Bc-SCC Biochain A501390 SMALL CELL CARCINOMA48-(84)-Bc-SCC Biochain A609167 SMALL CELL CARCINOMA 49-(85)-Bc-SCCBiochain A609169 SMALL CELL CARCINOMA 50-(33)-Bc-SCC Biochain A504115SMALL CELL CARCINOMA 51-As-N-PS Asterand 9078 Normal lung 52-As-N-PMAsterand 8757 Normal lung 53-As-N-PM Asterand 6692 Normal lung54-As-N-PM Asterand 7900 Normal lung 55-As-N-PM Asterand 8771 Normallung 56-As-N-PM Asterand 13094 Normal lung 57-As-N-PM Asterand 19174Normal lung 58-As-N-PM Asterand 13128 Normal lung 59-As-N-PM Asterand14374 Normal lung 60-(99)-Am-N PM Ambion 36856 Normal PM 61-(96)-Am-N PMAmbion 36853 Normal PM 62-(97)-Am-N PM Ambion 36854 Normal PM63-(93)-Am-N PM Ambion 111P0103A Normal PM 64-(98)-Am-N PM Ambion 36855Normal PM 69-(91)-Bc-N PM Biochain A607257 Normal (Pool 2) PM70-(90)-Bc-N PM Biochain A608152 Normal (Pool 2) PM

TABLE 4 Healthy panel RNA Details: sample name Source Sample id1-Bc-Rectum Biochain A610297 2-Bc-Rectum Biochain A610298 3-GC-Colon GCIZJ17R 4-GC-Colon GCI YUZNR 5-GC-Colon GCI 28QN6 6-Bc-Colon BiochainA501156 7-GC-Small bowel GCI V9L7D 8-Bc-Esoph Biochain A6038149-Bc-Esoph Biochain A603813 10-As-Panc Asterand 8918 11-As-Panc Asterand10082 12-As-Liver Asterand 7916 13-GC-Kidney GCI N1EVZ 14-GC-Kidney GCIBMI6W 15-Bc-Adrenal Biochain A610374 16-Am-Lung Ambion 111P0103A17-Bc-Lung Biochain A503205 18-As-Lung Asterand 6692 19-As-Lung Asterand7900 20-Am-Ovary Asterand 16848 21-GC-Ovary GCI Y9VHI 22-GC-Ovary GCIDD73B 23-GC-Ovary GCI FDPL9 24-GC-Cervix GCI E2P2N 25-ABS-Bladder ABS150300503 26-ABS-Bladder ABS 150700103 27-ABS-Bladder ABS 15070020328-Am-Placen Ambion 021P33A 29-Bc-Placen Biochain A411073 30-Am-BreastAmbion 26486 31-Am-Breast Ambion 23036 32-GC-Breast GCI E6UDD33-Bc-Breast Biochain A609234 34-Am-Prostate Ambion 25955 35-Bc-ProstateBiochain A609258 36-As-Testis Asterand 13071 37-As-Testis Asterand 1967138-TH-Blood-PBMC Tel- 52497 Hashomer 39-TH-Blood-PBMC Tel- 31055Hashomer 40-TH-Blood-PBMC Tel- 31058 Hashomer 41-Ic-Spleen IchilovCG-267 42-ABS-Spleen ABS 150800704 43-ABS-Spleen ABS 15080090444-ABS-Spleen ABS 150801804 45-ABS-Thymus ABS 13066 46-ABS-Thymus ABS13105 47-ABS-Thymus ABS 133968 48-Bc-Thyroid Biochain A61028749-Ic-Thyroid Ichilov CG-119-2 50-GC-Sali gland GCI NNSMV51-Ic-Cerebellum Ichilov CG-183-5 52-Bc-Brain Biochain A41132253-Bc-Brain Biochain A411079 54-ABS-Heart ABS 151101109 55-ABS-Heart ABS35208I026 56-ABS-Heart ABS 352JA02409 57-Ic-Heart (Fibrotic) IchilovCG-255-9 58-GC-Skel Mus GCI T8YZS 59-GC-Skel Mus GCI Q3WKA 60-As-SkelMus Asterand 8774 61-As-Skel Mus Asterand 10937 62-As-Skel Mus Asterand6692 63-ABS-Skin ABS 151104009 64-ABS-Skin ABS 352MC01909 65-ABS-SkinABS 150402309

TABLE 5 Kidney Panel RNA Details Sample Name Source Sample ID DiagnosisCancer Stage 1_AB_K_PM-N ABS ABS150303105 Alzheimer's 2_AB_K_PM-N ABSABS151200305 Alzheimer's 3_AB_K_PM-N ABS ABS151201805 Cardio VascularDisease 4_AB_K_PM-N ABS ABS24724672102 COPD 5_AB_K_RCC_ST2aN0MX ABSUH1003-29 RCC ST2aN0MX 7_AS_K_RCC_ST3aN0M1 Asterand 52813 (1066748F- RCCST3aN0M1 3152) 8_AS_K_RCC_ST3NXM1 Asterand 52819 (1066176F- RCC ST3NXM13152) 9_OR_K_RCC_ST4N1MX Origene CI0000011656 RCC ST4N1MX (1A26)10_OR_K_RCC_ST3aN0M1 Origene CU0000001623 RCC ST3aN0M1 (3714)11_OR_K_RCC_ST3N2M1 Origene CU0000009324 RCC ST3N2M1 (1A1A)12_OR_K_RCC_ST2NXMX Origene CX0000000190 RCC ST2NXMX (3D99)13_OR_K_RCC_ST2N0M1 Origene CU0000005834 RCC ST2N0M1 (34DD)14_OR_K_RCC_ST3bN0MX Origene CU0000000762 RCC ST3bN0MX (374D)15_OR_K_RCC_ST3NXMX Origene CI0000016503 RCC ST3NXMX (3743)16_OR_K_RCC_ST3aNXMX Origene CU0000001216 RCC ST3aNXMX (3711)17_AB_K_RCC_ST2N0MX ABS UH1002-14 RCC ST2N0MX 18_AB_K_RCC_ST2bN0M1 ABSUH1007-18 RCC ST2bN0M1 19_AB_K_PM-N ABS ABS150400105 ALS

TABLE 6 Liver Panel RNA Details Sample Name Source Sample ID DiagnosisCancer Stage 41_AB_L_PM-N ABS ABS151203707 Alzheimer's 42_AB_L_PM-N ABSABS151003509 Dementia 43_AS_L_PM-N Asterand 49874 (1143071F- Respiratoryarrest 3152) 44_AS_L_PM-N Asterand 50466 (1144029F- Unknown 3152)45_AS_L_PM-N Asterand 50483 (1144465F- Cardiopulmonary 3152) arrest46_AB_L_HCC_ST2N1MX ABS UH0603-43 HCC T2N1MX 47_AB_L_HCC_ST3N0MX ABSUH0901-55 HCC T3N0MX 48_AS_L_HCC_ST3N0M0 Asterand 51356 (1100251F- HCCT3N0M0 3152) 49_AS_L_HCC_ST4NXMX Asterand 51365 (1100271F- HCC T4NXMX3152) 50_AS_L_HCC_ST2N0M0 Asterand 52528 (1149074F- HCC T2N0M0 3152)51_OR_L_HCC_ST2N0MX Origene CI0000008358 HCC T2N0MX (1A25)52_OR_L_HCC_ST2NXMX Origene CI0000009200 HCC T2NXMX (14B1)53_OR_L_HCC_STXNXM1 Origene CI0000013002 HCC TXNXM1 (30B6)54_OR_L_HCC_ST3NXM1 Origene CI0000020838 HCC T3NXM1 (2445)55_OR_L_HCC_ST3NXMX Origene CU0000000996 HCC T3NXMX (15F6)56_OR_L_HCC_ST3NXMX Origene CU0000001197 HCC T3NXMX (02DE)57_OR_L_HCC_ST3NXMX Origene CI0000019267 HCC T3NXMX (2441)58_OR_L_HCC_ST2NXMX Origene CU0000005407 HCC T2NXMX (0F2D)59_OR_L_HCC_ST2NXMX Origene CU0000006675 HCC T2NXMX (0F2E)

TABLE 7 Housekeeping Genes For Rev HSKG primer primer Rev AmpliconAccession Seq seq For primer seq primer seq HSKG number ID ID sequenceID sequence id Amplicon sequence SDHA NM_004168 103 104 TGGGAA 105 CCACC106 TGGGAACAAG CAAGAG ACTGC AGGGCATCTG GGCATC ATCAA CTAAAGTTTC TG ATTCAAGATTCCATTT TG CTGCTCAGTAT CCAGTAGTGG ATCATGAATTT GATGCAGTGG TGG HPRT1NM_000194 107 108 TGACACT 109 GGTCCT 110 TGACACTGGCAA GGCAAAA TTTCACCAACAATGCAGAC CAATGCA AGCAAG TTTGCTTTCCTTG CT GTCAGGCAGTAT AATCCAAAGATGGTCAAGGTCGCA AGCTTGCTGGTGA AAAGGACC G6PD NM_000402 111 112 GAGGCCG 113GGACAG 114 GAGGCCGTCACC TCACCAA CCGGTC AAGAACATTCAC GAACAT AGAGCTCGAGTCCTGCATG AGCCAGATAGGC TGGAACCGCATC ATCGTGGAGAAG CCCTTCGGGAGGGACCTGCAGAGC TCTGACCGGCTGT CC UBC BC000449 133 134 ATTTGGG 135 TGCCTT136 ATTTGGGTCGCGG TCGCGGT GACATT TTCTTGTTTGTGG TCTTG CTCGATATCGCTGTGATCG GGT TCACTTGACAATG CAGATCTTCGTGA AGACTCTGACTG GTAAGACCATCACCCTCGAGGTTGA GCCCAGTGACAC CATCGAGAATGT CAAGGCA RPL19 NM_000981 119 120TGGCAAG 121 TGATCA 122 TGGCAAGAAGAA AAGAAGG GCCCAT GGTCTGGTTAGACTCTGGTTAG CTTTGAT CCCAATGAGACC GAG AATGAAATCGCC AATGCCAACTCCCGTCAGCAGATCC GGAAGCTCATCA AAGATGGGCTGA TCA PBGD BC019323 115 116 TGAGAGT117 CCAGGG 118 TGAGAGTGATTC GATTCGC TACGAG GCGTGGGTACCC GTGGG GCTTTCGCAAGAGCCAGC AAT TTGCTCGCATACA GACGGACAGTGT GGTGGCAACATT GAAAGCCTCGTACCCTGG

Specific primers and amplicons used for expression analysis of LSRtranscripts are provided in Table 8.

TABLE 8 LSR Primers and Amplicons For Rev Amplicon Forward primer ForReverse primer Rev Amplicon SEQ Amplicon primer SEQ primer primer SEQprimer name ID NO sequence name ID NO sequence name ID NO sequenceLSR_seg21-24_200- 137 GTCGACAAC LSR_seg21F_200-307 138 GTCGALSR_seg24R_200-308 139 AAGGT 307/308_Amplicon CAGCTCAAT CAACC CAGGTGCCCAGCTG AGCTC CAGCA GCAGCCGGG AATGC TTTCC AACCCAGGC TACAACCCC TACGTCGAGTGCCAG GACAGCGTG CGCACCGTC AGGGTCGTG GCCACCAAG CAGGGCAAC GCTGTGACCCTGGGA GATTACTAC CAGGGCCGG AGGATTACC ATCACCGGA AATGCTGAC CTGACC TTLSR_seg24-36_200- 140 ATGCTGACC LSR_seg24F_200-309 141 ATGCTLSR_seg36R_200-310 142 CCAGG 309/310_Amplicon TGACCTTTGA GACCT TAAGACCAGACGGC GACCT GCTCA GTGGGGGGA TTGAC GCCAC CAGTGGTGT GTATTACTGC TCCGTGGTCTCAG CCCAGGACC TCCAGGGGA ACAATGAGG CCTACGCAG AGCTCATCG TCCTTGGGAGGACCT CAGGGGTGG CTGAGCTCTT ACCTGG

Specific primers and amplicons used for expression analysis of TMEM25transcript is provided in Table 9.

TABLE 9 TMEM25 primers and amplicons Rev For primer  Amplicon Forwardprimer For SEQ Rev Amplicon SEQ Amplicon primer SEQ primer Reverse IDprimer name ID NO sequence name ID NO sequence primer name NO sequenceTMEM25_seg_21- 123 TTCACTGTCACT TMEM25_seg21F_200- 124 TTCATMEM25_seg27R_200- 125 CACC 27_200-344/ GCCCATCGGGCC 344 CTGT 346 AGGG346_Amplicon CAGCATGAGCTC CACT CAAA AACTGCTCTCTG GCCC CAGG CAGGACCCCAGAATCGG ACAAC AGTGGCCGATCA GCCAACGCCTCT GTCATCCTTAAT GTGCAATTCAAGCCAGAGATTGCC CAAGTCGGCGCC AAGTACCAGGAA GCTCAGGGCCCA GGCCTCCTGGTTGTCCTGTTTGCC CTGGTG

The expression data of LSR_seg24-36_200-309/310_Amplicon (SEQ ID:140) isdescribed in Examples 3-9 below.

Example 3

Expression of LSR_Transcripts which are Detectable by Amplicon asDepicted in Sequence Name LSR_Seg24-36_200-309/310 in Normal andCancerous Ovary Tissues

Expression of LSR transcripts detectable by or according toLSR_seg24-36_200-309/310_Amplicon (SEQ ID:140) and primersLSR_seg24F_200-309 (SEQ ID:141) and LSR_seg36R_200-310 (SEQ ID:142) wasmeasured by real time PCR. Non-detected samples (sample(s) no. 28) wereassigned Ct value of 41 and were calculated accordingly. In parallel theexpression of several housekeeping genes—SDHA (SEQ ID:103) (GenBankAccession No. NM_004168; amplicon—SDHA_Amplicon (seq ID: 106)), HPRT1(SEQ ID:107) (GenBank Accession No. NM_000194; HPRT1_Amplicon(SEQID:110)), and G6PD (SEQ ID:111) (GenBank Accession No. NM_000402;G6PD_Amplicon (SEQ ID:1114)) was measured similarly. For each RT sample,the expression of the above amplicon was normalized to the normalizationfactor calculated from the expression of these house keeping genes asdescribed in normalization method 2 in the “materials and methods”section. The normalized quantity of each RT sample was then divided bythe median of the quantities of the normal samples (sample numbers 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63 and 64, Table 1 above), to obtain a value of foldup-regulation for each sample relative to median of the normal samples.

FIG. 12 is a histogram showing over expression of the above-indicatedLSR transcripts in cancerous Ovary samples relative to the normalsamples.

As is evident from FIG. 12, the expression of LSR transcripts detectableby the above amplicon in serous carcinoma, mucinous carcinoma andadenocarcinoma samples was significantly higher than in thenon-cancerous samples (sample numbers 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 and 64, Table1 above). Notably an over-expression of at least 5 fold was found in 21out of 27 serous carcinoma samples, in 7 out of 9 mucinous carcinomasamples and in 7 out of 8 endometroid carcinoma samples.

Statistical analysis was applied to verify the significance of theseresults, as described below.

The P value for the difference in the expression levels of LSRtranscripts detectable by the above amplicon in Ovary serous carcinomasamples versus the normal tissue samples was determined by T test as2.22e-002. The P value for the difference in the expression levels ofLSR transcripts detectable by the above amplicon in Ovary mucinouscarcinoma samples versus the normal tissue samples was determined by Ttest as 6.84e-004. The P value for the difference in the expressionlevels of LSR transcripts detectable by the above amplicon in Ovaryendometroid carcinoma samples versus the normal tissue samples wasdetermined by T test as 4.61e-003. The P value for the difference in theexpression levels of LSR transcripts detectable by the above amplicon inOvary Adenocarcinoma samples versus the normal tissue samples wasdetermined by T test as 5.68e-004.

Threshold of 5 fold over expression was found to differentiate betweenserous carcinoma and normal samples with P value of 2.59e-009 as checkedby exact Fisher test. Threshold of 5 fold over expression was found todifferentiate between mucinous carcinoma and normal samples with P valueof 8.43e-006 as checked by exact Fisher test. Threshold of 5 fold overexpression was found to differentiate between endometroid carcinoma andnormal samples with P value of 2.38e-006 as checked by exact Fishertest. Threshold of 5 fold over expression was found to differentiatebetween Adenocarcinoma samples and normal samples with P value of7.28e-012 as checked by exact Fisher test.

The above values demonstrate statistical significance of the results.

Primer pairs are also optionally and preferably encompassed within thepresent invention; for example, for the above experiment, the followingprimer pair was used as a non-limiting illustrative example only of asuitable primer pair: LSR_seg24F_200-309 (SEQ ID:141); andLSR_seg36R_200-310 (SEQ ID:142).

The present invention also preferably encompasses any amplicon obtainedthrough the use of any suitable primer pair; for example, for the aboveexperiment, the following amplicon was obtained as a non-limitingillustrative example only of a suitable amplicon:LSR_seg24-36_200-309/310_Amplicon (SEQ ID:140).

Example 4

Expression of LSR_Transcripts which are Detectable by Amplicon asDepicted in Sequence Name LSR_Seg24-36_200-309/310 in Normal andCancerous Breast Tissues

Expression of LSR transcripts detectable by or according toseg24-36FR—LSR_seg24-36_200-309/310_Amplicon (SEQ ID:140) and primersLSR_seg24F_200-309 (SEQ ID:141) and LSR_seg36R_200-310 (SEQ ID:142) wasmeasured by real time PCR. Non-detected samples (sample(s) no. 81) wereassigned Ct value of 41 and were calculated accordingly. In parallel theexpression of several housekeeping genes—G6PD (SEQ ID:111) (GenBankAccession No. NM_000402; G6PD_Amplicon), PBGD (SEQ ID:115) (GenBankAccession No. BC019323; PBGD_Amplicon) RPL19 (SEQ ID:119) (GenBankAccession No. NM_000981 RPL19_Amplicon) and SDHA (SEQ ID:103) (GenBankAccession No. NM_004168 SDHA_Amplicon) was measured similarly. For eachRT sample, the expression of the above amplicon was normalized to thenormalization factor calculated from the expression of these housekeeping genes as described in normalization method 2 in the “materialsand methods” section. The normalized quantity of each RT sample was thendivided by the median of the quantities of the normal samples (samplenumbers 43, 45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 58, 59, 60, 61, 62,63, 64, 66, 67, 68 and 69, Table 2 above), to obtain a value of foldup-regulation for each sample relative to median of the normal samples.

FIG. 13 is a histogram showing over expression of the above-indicatedLSR transcripts in cancerous Breast samples relative to the normalsamples.

As is evident from FIG. 13, the expression of LSR transcripts detectableby the above amplicon in cancer samples was higher than in thenon-cancerous samples (sample numbers 43, 45, 46, 47, 48, 49, 50, 51,52, 54, 56, 58, 59, 60, 61, 62, 63, 64, 66, 67, 68 and 69, Table 2above). Notably an over-expression of at least 5 fold was found in 9 outof 53 adenocarcinoma samples.

Primer pairs are also optionally and preferably encompassed within thepresent invention; for example, for the above experiment, the followingprimer pair was used as a non-limiting illustrative example only of asuitable primer pair: LSR_seg24F_200-309 (SEQ ID: 141); andLSR_seg36R_200-310 (SEQ ID: 142).

The present invention also preferably encompasses any amplicon obtainedthrough the use of any suitable primer pair; for example, for the aboveexperiment, the following amplicon was obtained as a non-limitingillustrative example only of a suitable amplicon:LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140).

Example 5

Expression of LSR_Transcripts which are Detectable by Amplicon asDepicted in Sequence Name LSR_Seg24-36_200-309/310 in Normal andCancerous Lung Tissues

Expression of LSR transcripts detectable by or according to seg24-361-RLSR_seg24-36_200-309/310_Amplicon (SEQ ID:140) and primersLSR_seg24F_200-309 (SEQ ID:141) and LSR_seg36R_200-310 (SEQ ID:142) wasmeasured by real time PCR. In parallel the expression of severalhousekeeping genes—HPRT1(SEQ ID:107) (GenBank Accession No. NM_000194HPRT1_Amplicon(SEQ ID:110)), PBGD (SEQ ID:115) (GenBank Accession No.BC019323; PBGD_Amplicon(SEQ ID:118)), SDHA (SEQ ID:103) (GenBankAccession No. NM_004168; SDHA_Amplicon(SEQ ID:106)) and Ubiquitin (SEQID:133) (GenBank Accession No. BC000449; Ubiquitin_Amplicon(SEQ ID:136))was measured similarly. For each RT sample, the expression of the aboveamplicon was normalized to the normalization factor calculated from theexpression of these house keeping genes as described in normalizationmethod 2 in the “materials and methods” section. The normalized quantityof each RT sample was then divided by the median of the quantities ofthe normal samples (sample numbers 51, 52, 53, 54, 56, 57, 58, 59, 60,61, 62, 63, 64, 69 and 70, Table 3 above), to obtain a value of foldup-regulation for each sample relative to median of the normal samples.

FIG. 14 is a histogram showing over expression of the above-indicatedLSR transcripts in cancerous Lung samples relative to the normalsamples.

As is evident from FIG. 14, the expression of LSR or transcriptsdetectable by the above amplicon in adenocarcinoma and non-small cellcarcinoma samples was significantly higher than in the non-canceroussamples (sample numbers 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63,64, 69 and 70, Table 3 above) and was higher in a few squamous cellcarcinoma samples than in the non-cancerous samples. Notably anover-expression of at least 5 fold was found in 7 out of 15adenocarcinoma samples, in 3 out of 18 squamous cell carcinoma samplesand in 10 out of 40 non-small cell carcinoma samples.

Statistical analysis was applied to verify the significance of theseresults, as described below.

The P value for the difference in the expression levels of Homo sapienslipolysis stimulated lipoprotein receptor transcripts detectable by theabove amplicon in Lung adenocarcinoma samples versus the normal tissuesamples was determined by T test as 2.98e-005. The P value for thedifference in the expression levels of LSR transcripts detectable by theabove amplicon in Lung squamous cell carcinoma samples versus the normaltissue samples was determined by T test as 7.42e-003. The P value forthe difference in the expression levels of Homo sapiens lipolysisstimulated lipoprotein receptor transcripts detectable by the aboveamplicon in Lung large cell carcinoma samples versus the normal tissuesamples was determined by T test as 1.76e-002. The P value for thedifference in the expression levels of Homo sapiens lipolysis stimulatedlipoprotein receptor transcripts detectable by the above amplicon inLung small cell carcinoma samples versus the normal tissue samples wasdetermined by T test as 4.35e-002. The P value for the difference in theexpression levels of Homo sapiens lipolysis stimulated lipoproteinreceptor transcripts detectable by the above amplicon in Lung non-smallcell carcinoma samples versus the normal tissue samples was determinedby T test as 4.31e-006.

Threshold of 5 fold over expression was found to differentiate betweenadenocarcinoma and normal samples with P value of 3.16e-003 as checkedby exact Fisher test. Threshold of 5 fold over expression was found todifferentiate between non-small cell carcinoma and normal samples with Pvalue of 2.90e-002 as checked by exact Fisher test.

The above values demonstrate statistical significance of the results.

Primer pairs are also optionally and preferably encompassed within thepresent invention; for example, for the above experiment, the followingprimer pair was used as a non-limiting illustrative example only of asuitable primer pair: LSR_seg24F_200-309 (SEQ ID: 141).; andLSR_seg36R_200-310 (SEQ ID: 142).

The present invention also preferably encompasses any amplicon obtainedthrough the use of any suitable primer pair; for example, for the aboveexperiment, the following amplicon was obtained as a non-limitingillustrative example only of a suitable amplicon:LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140).

Example 6

Expression of LSR_Transcripts which are Detectable by Amplicon asDepicted in Sequence Name LSR_Seg24-36_200-309/310 in Different NormalTissues

Expression of LSR transcripts detectable by or according to seg24-361-RLSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) and primersLSR_seg24F_200-309 (SEQ ID:141) and LSR_seg36R_200-310 (SEQ ID: 142) wasmeasured by real time PCR. In parallel the expression of severalhousekeeping genes—SDHA (SEQ ID:103) (GenBank Accession No. NM_004168;SDHA_Amplicon (SEQ ID:106)), HPRT1 (SEQ ID:107) (GenBank Accession No.NM_000194; HPRT1_Amplicon(SEQ ID:110)), and G6PD (SEQ ID:111) (GenBankAccession No. NM_000402; G6PD_Amplicon (SEQ ID:114)) was measuredsimilarly. For each RT sample, the expression of the above amplicon wasnormalized to the normalization factor calculated from the expression ofthese house keeping genes as described in normalization method 2 in the“materials and methods” section. The normalized quantity of each RTsample was then divided by the median of the quantities of the Ovarysamples (sample numbers 20, 21, 22 and 23, Table 4 above), to obtain avalue of relative expression of each sample relative to median of theOvary samples.

FIG. 15 is a histogram showing the expression of the above-indicated LSRtranscripts in normal tissue samples relative to the ovary samples.

Example 7

Expression of LSR_Transcripts which are Detectable by Amplicon asDepicted in Sequence Name LSR_Seg24-36_200-309/310 in Normal andCancerous Kidney Tissues

Expression of LSR transcripts detectable by or according toseg24-36FR—LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) and primersLSR_seg24F_200-309 (SEQ ID: 141) and LSR_seg36R_200-310 (SEQ ID: 142)was measured by real time PCR. In parallel the expression of severalhousekeeping genes—SDHA (SEQ ID:103) (GenBank Accession No. NM_004168;SDHA_Amplicon (SEQ ID:106)), G6PD (SEQ ID:111) (GenBank Accession No.NM_000402; G6PD_Amplicon (SEQ ID:114)) and PBGD (SEQ ID:115) (GenBankAccession No. BC019323; PBGD_Amplicon(SEQ ID:118)) was measuredsimilarly. For each RT sample, the expression of the above amplicon wasnormalized to the normalization factor calculated from the expression ofthese house keeping genes as described in normalization method 2 in the“materials and methods” section. The normalized quantity of each RTsample was then divided by the median of the quantities of the normalsamples (sample numbers 1, 2, 3, 4 and 19, Table 5 above), to obtain avalue of fold up-regulation for each sample relative to median of thenormal samples.

FIG. 16 is a histogram showing down regulation of the above-indicatedHomo sapiens lipolysis stimulated lipoprotein receptor transcripts incancerous Kidney samples relative to the normal samples.

As is evident from FIG. 16, the expression of LSR transcripts detectableby the above amplicon in cancerous Kidney samples was significantlylower than in the non-cancerous samples (sample numbers 1, 2, 3, 4 and19, Table 5 above).

Statistical analysis was applied to verify the significance of theseresults, as described below.

The P value for the difference in the expression levels of LSRtranscripts detectable by the above amplicon in cancerous Kidney samplesversus the normal tissue samples was determined by T test as 1.25e-01.

Primer pairs are also optionally and preferably encompassed within thepresent invention; for example, for the above experiment, the followingprimer pair was used as a non-limiting illustrative example only of asuitable primer pair: LSR_seg24F_200-309 (SEQ ID: 141); andLSR_seg36R_200-310 (SEQ ID: 142).

The present invention also preferably encompasses any amplicon obtainedthrough the use of any suitable primer pair; for example, for the aboveexperiment, the following amplicon was obtained as a non-limitingillustrative example only of a suitable_seg24-36_200-309/310_Amplicon(SEQ ID:140).

Example 8

Expression of LSR_Transcripts which are Detectable by Amplicon asDepicted in Sequence Name LSR_Seg24-36_200-309/310 in Normal andCancerous Liver Tissues

Expression of LSR transcripts detectable by or according toseg24-36FR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) and primersLSR_seg24F_200-309 (SEQ ID: 141) and LSR_seg36R_200-310 (SEQ ID: 142)was measured by real time PCR. In parallel the expression of severalhousekeeping genes—SDHA (SEQ ID:103) (GenBank Accession No. NM_004168;SDHA_Amplicon (SEQ ID: 106)), G6PD (SEQ ID:111) (GenBank Accession No.NM_000402; G6PD_Amplicon (seq ID:114)) and PBGD (SEQ ID: 115) (GenBankAccession No. BC019323;—PBGD_Amplicon(SEQ ID:118)) was measuredsimilarly. For each RT sample, the expression of the above amplicon wasnormalized to the normalization factor calculated from the expression ofthese house keeping genes as described in normalization method 2 in the“materials and methods” section. The normalized quantity of each RTsample was then divided by the median of the quantities of the normalsamples (sample numbers 41, 42, 43, 44 and 45, Table 6 above), to obtaina value of fold up-regulation for each sample relative to median of thenormal samples.

FIG. 17 is a histogram showing the expression of the above-indicated LSRtranscripts in cancerous Liver samples relative to the normal samples.

Primer pairs are also optionally and preferably encompassed within thepresent invention; for example, for the above experiment, the followingprimer pair was used as a non-limiting illustrative example only of asuitable primer pair: LSR_seg24F_200-309 (SEQ ID: 141); andLSR_seg36R_200-310 (SEQ ID: 142) reverse primer.

The present invention also preferably encompasses any amplicon obtainedthrough the use of any suitable primer pair; for example, for the aboveexperiment, the following amplicon was obtained as a non-limitingillustrative example only of a suitable amplicon:LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140).

Example 9

Cloning of LSR_T1_P5a ORF Fused to Flag Tag

Cloning of LSR_T1_P5a open reading frame (ORF) (SEQ ID NO 154) fused toFLAG (amino acid sequence: DYKDDDDK, SEQ ID NO: 153) to generatedLSR_P5a protein (SEQ ID NO: 11) fused to flag, was performed by PCR asdescribed below.

A 3-step PCR reaction was performed using PfuUltra II Fusion HS DNAPolymerase (Agilent, Catalog no. 600670) under the following conditions:on the first step, 1 μl of undiluted Ovary sample (ID PZQXH) from theOvary panel (Table 1) served as a template for a PCR reaction with 0.5μl of each of the primers 200_369_LSR_Kozak_NheI (SEQ ID NO: 147) and200_379_LSR_Rev (SEQ ID NO: 148) in a total reaction volume of 25 μl.The reaction conditions were 5 minutes at 98° C.; 35 cycles of: 20seconds at 98° C., 30 seconds at 55° C. and 1.5 minutes at 72° C.; then5 minutes at 72° C. The PCR product was diluted 1:20 in DDW and 1 ul wasused as a template for each of the PCR reactions on step 2.

For the second step the 5′ part of LSR was amplified with 0.5 ul of eachof the primers 200_369_LSR_Kozak_NheI (10 μM) (SEQ ID NO: 147) and200_371_LSR_seg36R (10 uM) (SEQ ID NO: 149) in a total reaction volumeof 25 μl . The 3′ part of LSR was amplified with 0.5 ul of each of theprimers 200_370_LSR_seg36F (10 μM) (SEQ ID NO: 150) and200-373_LSR_Flag_BamHI_Rev (10 uM) (SEQ ID NO: 151) in a total reactionvolume of 25 μl .The reaction conditions for both reactions were 5minutes at 98° C.; 35 cycles of: 20 seconds at 98° C., 15 seconds at 60°C. and 1.5 minutes at 72° C.; then 5 minutes at 72° C. The products ofeach of the reactions were separated on 1% agarose gel and purified fromthe gel using Qiaquick™ Gel Extraction Kit (Qiagene, Catalog no. 28706).100 ng of the 5′ product and 100 ng of the 3′ product were used as atemplate for the third step of the PCR reaction, in which the fullLSR-Flag sequence was amplified. 0.5 μl of each of the primers200_369_LSR_Kozak_NheI (SEQ ID NO: 147) and 200-373_LSR_Flag_BamHI_Rev(SEQ ID NO: 151) in a total reaction volume of 25 μl . The reactionconditions were 5 minutes at 98° C.; 35 cycles of: 20 seconds at 98° C.,30 seconds at 55° C. and 1.5 minutes at 72° C.; then 5 minutes at 72° C.All of the primers that were used include gene specific sequences,restriction enzyme sites, Kozak sequence and FLAG tag sequence. The PCRproduct of step 3 was separated on 1% agarose gel. After verification ofthe expected band size, the PCR product was purified using QIAquick™ GelExtraction kit.

The purified full length PCR product was digested with NheI and BamHIrestriction enzymes (New England Biolabs, Beverly, Mass., U.S.A.). Afterdigestion, the DNA was separated on a 1% agarose gel. The expected bandsize was excised and extracted from the gel as described above. Thedigested DNA was then ligated into pIRESpuro3 vector that was digestedwith NheI and BamHI as described above, treated with AntarcticPhosphatase (New England Biolabs, Beverly, Mass., U.S.A., Catalog no.M0289L) for 30 minutes at 37° C. and purified from 1% agarose gel usingQIAquick™ Gel Extraction kit as described above. The ligation reactionwas performed with T4 DNA Ligase (Promega; Catalog no. M180A).

Example 10

Cloning of LSR_T1_P5a ORF

Cloning of LSR_T1_P5a open reading frame (ORF) (SEQ ID NO: 154) wasperformed by PCR to generate LSR_P5a protein (SEQ ID NO: 11), asdescribed below.

A PCR reaction was performed using PfuUltra II Fusion HS DNA Polymerase(Agilent, Catalog no. 600670) under the following conditions: 50 ng ofpIRES_puro3_LSR_T1_P5a_Flag construct described above served as atemplate for a PCR reaction with 0.5microliter of each of the primers200_369_LSR_Kozak_NheI (SEQ ID NO: 147) and 200-372_LSR_BamHI_Rev (SEQID NO: 152) in a total reaction volume of 25 μl . The reactionconditions were 5 minutes at 98° C.; 35 cycles of: 20 seconds at 98° C.,30 seconds at 55° C. and 1.5 minutes at 72° C.; then 10 minutes at 72°C. All of the primers that were used include gene specific sequences,restriction enzyme sites and Kozak sequence. The PCR product wasseparated on 1% agarose gel. After verification of the expected bandsize, the PCR product was purified using QIAquick™ Gel Extraction kit asdescribed above.

The purified PCR product was digested with NheI and BamHI restrictionenzymes (New England Biolabs, Beverly, Mass., U.S.A.). After digestion,the DNA was separated on a 1% agarose gel. The expected band size wasexcised and extracted from the gel as described above. The digested DNAwas then ligated into pIRESpuro3 vector that was digested with NheI andBamHI as described above, incubated with Antarctic Phosphatase (NewEngland Biolabs, Beverly, Mass., U.S.A., Catalog no. M0289L) for 30minutes at 37° C. and purified from 1% agarose gel using QIAquick™ GelExtraction kit as described above. The ligation reaction was performedwith T4 DNA Ligase (Promega; Catalog no. M180A).

Sequence verification of both tagged and untagged constructs describedabove was performed (Hylabs, Rehovot, Israel). Two nucleotides mismacheswere identified, as follows: G to A at nucleic acid position 119 of SEQID NO: 154, and A to G at nucleic acid position 626 from SEQ ID NO: 154,resulting in a nucleic sequence set forth in SEQ ID NO: 145 for theuntagged construct, and SEQ ID NO: 146 for the tagged construct;yielding a polypeptide having an amino acid mismatch I to M in aminoacid position 209 in SEQ ID NO:301, resulting in a protein having aminoacid sequence set forth in SEQ ID NO: 143 for the untagged construct andSEQ ID NO: 144 for the tagged construct.

The above recombinant plasmids were processed for stable pool generationas described below.

Example 11

Establishment of a Stable Pool of Recombinant HEK293T Cells ExpressingLSR_P5a_Flag_M Protein

HEK-293T cells were stably transfected with LSR_T1_P5a_Flag_m (SEQ IDNO: 146) and pIRESpuro3 empty vector plasmids as follows:

HEK-293T (ATCC, CRL-11268) cells were plated in a sterile 6 well platesuitable for tissue culture, containing 2 ml pre-warmed of completemedia, DMEM [Dulbecco's modified Eagle's Media, Biological Industries(Beit Ha'Emek, Israel, catalog number: 01-055-1A)+10% FBS [Fetal BovineSerum, Biological Industries (Beit Ha'Emek, Israel, catalog number:04-001-1A)+4 mM L-Glutamine (Biological Industries (Beit Ha'Emek,Israel), catalog number: 03-020-1A). 500,000 cells per well weretransfected with 2 μg of DNA construct using 6 μl FuGENE 6 reagent(Roche, catalog number: 11-814-443-001) diluted into 94 ul DMEM. Themixture was incubated at room temperature for 15 minutes. The complexmixture was added dropwise to the cells. The cells were placed in anincubator maintained at 37° C. with 5% CO2 content. 48 hours after thetransfection, the cells were transferred to a 75 cm2 tissue cultureflask containing 15 ml of selection medium: complete medium supplementedwith 5 μg\ml puromycin (Sigma, catalog number P8833). Cells were placedin an incubator, and the medium was replaced every 3-4 days, until cloneformation was observed.

Example 12

Analysis of the Ectopic Expression of LSR_P5a_Flag_M inStably-Transfected HEK293T Cells

The expression of LSR_P5a_Flag_m (SEQ ID NO 144) in stably-transfectedHEK293T cells was determined by Western blot analysis of the celllysates, using anti LSR Antibodies and anti flag antibody as indicatedin Table 9.

Cells were dissociated from the plate using Cell Dissociation BufferEnzyme-Free PBS-Based (Gibco; 13151-014), washed in Dulbecco's PhosphateBuffered Saline (PBS) (Biological Industries, 02*023-1A) and centrifugedat 1200 g for 5 minutes. Whole cell extraction was performed byresuspending the cells in 50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA,1% Triton X-100, supplemented with 25× complete EDTA free proteaseinhibitor cocktail (Roche, 11 873 580 001) and vortexing for 20 seconds.Cell extracts were collected following centrifugation at 20000 g for 20minutes at 4° C. and protein concentration was determined with BradfordBiorad Protein Assay (Biorad cat#500-0006). Equal protein amounts wereanalyzed by SDS-PAGE (Invitrogen NuPAGE 4-12% NuPAGE Bis Tris, Cat#NP0335, NP0322) and transferred to Nitrocellulose membrane (BA83, 0.2μm, Schleicher & Schuell, Cat#401385). The membrane was blocked withTTBS (Biolab, Cat#: 20892323)/10% skim milk (Difco, Cat#232100) andincubated with the indicated primary antibodies (FIG. 18) diluted inTTBS/5% BSA (Sigma-Aldrich, A4503) at the indicated concentrations(Table 9), for 16 hours at 4° C. After 3 washes with TTBS, The membranewas further incubated for 1 hour at Room Temperature with thesecondary-conjugated antibodies as indicated, diluted in TTBS.Chemiluminescence reaction was performed with ECL Western BlottingDetection Reagents (GE Healthcare, Cat # RPN2209) and the membrane wasexposed to Super RX Fuji X-Ray film (Catalog no. 4741008389).

FIG. 18 demonstrates the expression of LSR_P5a_Flag_m protein (SEQ ID:144) in recombinant HEK293T cells at the expected band size ˜70 kDa, asdetected with anti Flag (Sigma cat#A8592) (FIG. 18A) and anti LSRantibodies as follow: Abnova, cat#H00051599-B01P (FIG. 18B) Abcam, catab59646 (FIG. 18C) and Sigma cat# HPA007270 (FIG. 18D).

Example 13

Determination of the Subcellular Localization of the EctopicLSR_P5a_Flag_M in HEK293T Cells

The subcellular localization of the LSR_P5a_Flag_m protein (SEQ ID NO:144) was determined in stably-transfected cells by confocal microscopy.

Stably transfected recombinant HEK293T cells expressing a LSR_P5a_Flag_m(SEQ ID NO: 144) described above were plated on coverslips pre-coatedwith Poly-L-Lysine (Sigma; Catalogue no. P4832). After 24 hrs the cellswere processed for immunostaining and analyzed by confocal microscopy.The cover slip was washed in phosphate buffered saline (PBS), then fixedfor 15 minutes in a solution of PBS/3.7% paraformaldehyde (PFA) (EMS,catalog number: 15710)/3% glucose (Sigma, catalog number: G5767). ThePFA was Quenched with PBS/3 mM Glycine (Sigma, catalog number: G7126)for 5 minutes. After two 5-minute washes in PBS, the cells werepermeabilized with PBS/0.1% Triton-X100 for 5 minutes at RoomTemperature and wash twice in PBS. Then, blocking of non-specificregions was performed with PBS/5% Bovine Serum Albumin (BSA) (Sigma,catalog number: A4503) for 20 minutes. The coverslip was then incubatedin a humid chamber for 1 hour with each of the primary antibodiesantibodies diluted in PBS/5% BSA as indicated, followed by three5-minute washes in PBS. The coverslips were then incubated for 30minutes with the corresponding secondary antibody diluted in PBS/2.5%BSA at the indicated dilution. The antibodies and the dilutions thatwere used are specified in Table 9. After a prewash in Hank's BalancedSalt Solutions w/o phenol red (HBSS) (Biological Industries Catalog no.02-016-1), the coverslip was incubated with WGA-Alexa 488 (Invitrogen,catalog number W11261) diluted 1:200 in HBSS for 10 min, washed in HBSSand incubated in BISBENZIMIDE H 33258 (Sigma, catalog number: 14530)diluted 1:1000 in HBSS. The coverslip was then mounted on a slide withGel Mount Aqueous medium (Sigma, catalog number: G0918) and cells wereobserved for the presence of fluorescent product using confocalmicroscopy.

The subcellular localization of LSR_P5a_Flag_m is demonstrated in FIG.19, LSR_P5a_Flag_m (SEQ ID NO: 144) is localized mainly to the cellcytoplasm, but can also be detected on the cell surface as detected withanti Flag (Sigma cat# A9594) (FIG. 19A) and anti LSR antibodies asfollows: Abcam,cat ab59646 (FIG. 19B) Abnova, cat#H00051599-B01P (FIG.19C) and Sigma cat# HPA007270 (FIG. 19D).

Example 14 Analysis of the Expression of Endogenous LSR Protein inVarious Cell Lines

The expression of endogenous LSR protein in various cell lines wasanalyzed by Western Blotting as described below.

SK-OV3 (ATCC no. HTB-77) Caov3 (ATCC no. HTB-75), OVCAR3 (ATCC no.HTB-161), ES-2 (ATCC no. CRL-1978), OV-90 (ATCC no. CRL-11732) TOV112D(ATCC no. CRL-11731) and Hep G2 (ATCC no. HB-8065) cell extracts wereprepared as described above.

HeLa (catalog no. sc-2200), MCF-7 (catalog no. sc-2206), CaCo2 (catalogno. sc-2262) and SkBR3 (catalog no. sc-2218) cell extracts werepurchased from SantaCruz Biotechnology.

Equal protein amounts were analyzed by SDS-PAGE and transferred toNitrocellulose membrane as described above. The membrane was blockedwith TTBS (Biolab, Cat#: 20892323)/10% skim milk (Difco, Cat#232100) andincubated with anti LSR antibodies (Abcam,cat#ab59646) diluted inTTBS/5% BSA (Sigma-Aldrich, A4503) at the indicated concentrations(Table 9), for 16 hours at 4° C. After 3 washes with in TTBS, Themembrane was further incubated for 1 hour at Room Temperature with thesecondary-conjugated antibodies as indicated (Table 9), diluted in TTBS.Chemiluminescence reaction was performed with ECL Western BlottingDetection Reagents (GE Healthcare, Cat # RPN2209) and the membrane wasexposed to Super RX Fuji X-Ray film (Catalog no. 4741008389).

FIG. 20 demonstrates the endogenous expression of LSR in various celllines. A band at 72 kDa corresponding to LSR was detected with anti LSRantibody in extracts of SK-OV3, Caov3, OVCAR3, OV-90, Hep G2, HeLa,CaCo2, and SkBR3 (FIG. 20A). Anti GAPDH (Abcam cat# ab9484) served as aloading control (FIG. 20B).

TABLE 9 Primary and secondary antibodies Antibody Application DilutionMouse Anti FLAG-Cy3 (Sigma catalog IF 1:200 number: A9594) Mouse AntiFLAG-HRP (Sigma Catalog no. WB  1:2000 A8592) Rabbit Anti LSR (Abcamcatalog number: IF 1:500 ab59646) WB  1:4000 Rabbit Anti LSR (Sigmacatalog number: IF 1:100 HPA007270) WB  1:2500 Mouse Anti LSR (Abnovacatalog number: IF 1:500 H00051599-B01P) WB  1:1000 Mouse Anti GAPDH(Abcam catalog number: WB  1:1000 ab9484) Donkey Anti Rabbit Cy3(Jackson IF 1:200 ImmunoResearch Laboratories Inc. catalog no.711-165-152) Donkey Anti Mouse Dylight 549 (Jackson IF 1:100ImmunoResearch Laboratories Inc. catalog no. 715-506-150) Peroxidaseconjugated affinity purified Goat WB  1:10000 Anti Rabbit IgG (JacksonImmunoResearch Laboratories Inc. catalog no. 111-035-003) Peroxidaseconjugated affinity purified Goat WB  1:10000 Anti-Mouse IgG (JacksonImmunoResearch Laboratories Inc. catalog no. 115-035-146)

Example 15

Expression of TMEM25_Transcripts which are Detectable by Amplicon asDepicted in Sequence Name TMEM25_Seg21-27 in Normal and Cancerous BreastTissues

Expression of TMEM25 transcripts detectable by or according toseg21-27-TMEM25_seg_21-27_200-344/346_Amplicon (SEQ ID NO: 123) andprimers TMEM25_seg21F_200-344 (SEQ ID NO.124) and TMEM25_seg27R_200-346(SEQ ID NO.125) was measured by real time PCR. In parallel theexpression of several housekeeping genes—G6PD (GenBank Accession No.NM_000402; (SEQ ID NO.111) G6PD_Amplicon (SEQ ID NO.114)), RPL19(GenBank Accession No. NM_000981; (SEQ ID NO.119)—RPL19_Amplicon (SEQ IDNO.122)), PBGD (GenBank Accession No. BC019323; (SEQ ID NO.115)PBGD_Amplicon (SEQ ID NO.118)) and SDHA (GenBank Accession No.NM_004168; (SEQ ID NO.103) SDHA_Amplicon (SEQ ID NO.106)) was measuredsimilarly. For each RT sample, the expression of the above amplicon wasnormalized to the normalization factor calculated from the expression ofthese house keeping genes as described in “materials and methods”section. The normalized quantity of each RT sample was then divided bythe median of the quantities of the normal samples (sample numbers 43,45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, 66,67, 68 and 69, Table 1 above), to obtain a value of fold differentialexpression for each sample relative to median of the normal samples.

In two experiments that were carried out no differential expression inthe cancerous samples relative to the normal samples was observed (FIG.21).

Primer pairs are also optionally and preferably encompassed within thepresent invention; for example, for the above experiment, the followingprimer pair was used as a non-limiting illustrative example only of asuitable primer pair: TMEM25_seg21F_200-344 (SEQ ID NO.124) forwardprimer; and TMEM25_seg27R_200-346 (SEQ ID NO.125) reverse primer.

The present invention also preferably encompasses any amplicon obtainedthrough the use of any suitable primer pair; for example, for the aboveexperiment, the following amplicon was obtained as a non-limitingillustrative example only of a suitable amplicon:TMEM25_seg_21-27_200-344/346_Amplicon (SEQ ID NO: 123).

Example 16

Expression of TMEM25_Transcripts which are Detectable by Amplicon asDepicted in Sequence Name TMEM25 Seg21-27 in Different Normal Tissues

Expression of TMEM25 transcripts detectable by or according toseg21-27-TMEM25_seg_21-27_200-344/346_Amplicon (SEQ ID NO: 123) andprimers TMEM25_seg21F_200-344 (SEQ ID NO.124) and TMEM25_seg27R_200-346(SEQ ID NO.125) was measured by real time PCR. In parallel theexpression of several housekeeping genes—SDHA (GenBank Accession No.NM_004168; (SEQ ID NO.103) SDHA_Amplicon (SEQ ID NO.106)), G6PD (GenBankAccession No. NM_000402; (SEQ ID NO.111) G6PD_Amplicon (SEQ ID NO.114))and HPRT1 (GenBank Accession No. NM_000194; (SEQ ID NO.107)HPRT1_Amplicon (SEQ ID NO.110)) were measured similarly. For each RTsample, the expression of the above amplicon was normalized to thenormalization factor calculated from the expression of these housekeeping genes as described in normalization method 2 in the “materialsand methods” section. The normalized quantity of each RT sample was thendivided by the median of the quantities of the Breast samples (samplenumbers 30, 31, 32 and 33, Table 2 above), to obtain a value of relativeexpression of each sample relative to median of the Breast samples (FIG.22).

Example 17 Cloning of TMEM25 Proteins

Cloning of TMEM25_T0_P5 ORF Fused to Flag Tag

Cloning of TMEM25_T0_P5 open reading frame (ORF) (SEQ ID NO: 130) fusedto FLAG (SEQ ID NO: 153) was carried out by RT PCR as described below.

1 μl of undiluted Colon cancer pool DNA served as a template for a PCRreaction. The PCR was done using KAPA Hifi DNA polymerase(KAPABIOSYSTEM, Catalog no. KK2101) under the following conditions: 1μl—cDNA described above; 1 μl (25 μM)—of each primer200-374_TMEM25_NheI_Kozak_seg5F (SEQ ID NO: 127) and200-375_TMEM25_Flag_STOP_EcoRI_seg43R (SEQ ID NO: 128) in a totalreaction volume of 50 μl ; with a reaction program of 5 minutes in 95°C.; 40 cycles of: 20 seconds at 98° C., 15 seconds at 55° C., 1 minuteat 72° C.; then 5 minutes at 72° C. Primers which were used include genespecific sequences; restriction enzyme sites; Kozak sequence and FLAGtag.

25 μl of PCR product were loaded onto a 1.5% agarose gel stained withethidium bromide, electrophoresed in 1×TAE solution at 100V, andvisualized with UV light. After verification of expected band size. 1 μlof the PCR product above template were served as a template forreamplification. The PCR was done using KAPA Hifi DNA polymerase(KAPABIOSYSTEM, Catalog no. KK2101) under the same conditions describedabove.

PCR product was purified from gel using QIAquick™ Gel Extraction kit(Qiagen, catalog number: 28707).

The purified PCR product was digested with NheI and EcoRI restrictionenzymes (New England Biolabs, Beverly, Mass., U.S.A.). The digested DNAwas then ligated into pIRESpuro3 (pRp) vector (Clontech, cat No: 631619)previously digested with the above restriction enzymes, using T4 DNAligase (Promega, catalog number: M1801). The resulting DNA wastransformed into competent E. Coli bacteria DH5a (RBC Bioscience,Taipei, Taiwan, catalog number: RH816) according to manufacturer'sinstructions, then plated on LB-ampicillin agar plates for selection ofrecombinant plasmids, and incubated overnight at 37° C. The followingday, positive colonies were screened by PCR using pIRESpuro3 vectorspecific primer and gene specific primer (data not shown). The PCRproduct was analyzed using 2% agarose gel as described above. Afterverification of expected band size, positive colonies were grown in 5 mlTerrific Broth supplemented with 100 μg/ml ampicillin, with shakingovernight at 37° C. Plasmid DNA was isolated from bacterial culturesusing Qiaprep™ Spin Miniprep Kit (Qiagen, catalog number: 27106).Accurate cloning was verified by sequencing the inserts (Hylabs,Rehovot, Israel). Upon verification of an error-free colony (i.e. nomutations within the ORF), recombinant plasmids were processed forfurther analyses.

Cloning of TMEM25_T0_P5 ORF Non Tagged

Cloning of TMEM25_T0_P5 open reading frame (ORF) non tagged (SEQ ID NO:130) was carried out by RT PCR as described below.

1 μl of undiluted Colon cancer pool DNA served as a template for a PCRreaction. The PCR was done using KAPA Hifi DNA polymerase(KAPABIOSYSTEM, Catalog no. KK2101) under the following conditions: 1μl—cDNA described above; 1 μl (25 μM)—of each primer200-374_TMEM25_NheI_Kozak_seg5F (SEQ ID NO: 127) and200-377_TMEM25_STOP_EcoRI_seg43R (SEQ ID NO: 131) in a total reactionvolume of 50 μl ; with a reaction program of 5 minutes in 95° C.; 40cycles of: 20 seconds at 98° C., 15 seconds at 55° C., 1 minute at 72°C.; then 5 minutes at 72° C. Primers which were used include genespecific sequences; restriction enzyme sites and Kozak sequence.

25 μl of PCR product were loaded onto a 1.5% agarose gel stained withethidium bromide, electrophoresed in 1×TAE solution at 100V, andvisualized with UV light. After verification of expected band size. 5 μlof the PCR product above template were served as a template forreamplification. The PCR was done using KAPA Hifi DNA polymerase(KAPABIOSYSTEM, Catalog no. KK2101) under the same conditions describedabove.

PCR product was purified from gel using QIAquick™ Gel Extraction kit(Qiagen, catalog number: 28707).

The purified PCR product was digested with NheI and EcoRI restrictionenzymes (New England Biolabs, Beverly, Mass., U.S.A.). The digested DNAwas then ligated into pIRESpuro3 (pRp) vector (Clontech, cat No: 631619)previously digested with the above restriction enzymes, using T4 DNAligase (Promega, catalog number: M1801). The resulting DNA wastransformed into competent E. Coli bacteria DH5a (RBC Bioscience,Taipei, Taiwan, catalog number: RH816) according to manufacturer'sinstructions, then plated on LB-ampicillin agar plates for selection ofrecombinant plasmids, and incubated overnight at 37° C. The followingday, positive colonies were screened by PCR using pIRESpuro3 vectorspecific primer and gene specific primer (data not shown). The PCRproduct was analyzed using 2% agarose gel as described above. Afterverification of expected band size, positive colonies were grown in 5 mlTerrific Broth supplemented with 100 μg/ml ampicillin, with shakingovernight at 37° C. Plasmid DNA was isolated from bacterial culturesusing Qiaprep™ Spin Miniprep Kit (Qiagen, catalog number: 27106).Accurate cloning was verified by sequencing the inserts (Hylabs,Rehovot, Israel). Upon verification of an error-free colony (i.e. nomutations within the ORF), recombinant plasmids were processed forfurther analyses.

Example 18

Generation of Stable Pool Expressing TMEM25_(—) P5 and TMEM25_(—)P5_Flag Proteins

The TMEM25_T0_P5 (SEQ ID NO: 130) and TMEM25_T0_P5_FLAG (SEQ ID NO: 126)pIRESpuro3 constructs or pIRESpuro3 empty vector were stably transfectedinto HEK-293T cells as follows:

HEK-293T (ATCC, CRL-11268) cells were plated in a sterile 6 well platesuitable for tissue culture, using 2 ml pre-warmed of complete media,DMEM [Dulbecco's modified Eagle's Media, Biological Industries (BeitHa'Emek, Israel, catalog number: 01-055-1A)+10% FBS [Fetal Bovine Serum,Biological Industries (Beit Ha'Emek, Israel, catalog number:04-001-1A)+4 mM L-Glutamine (Biological Industries (Beit Ha'Emek,Israel), catalog number: 03-020-1A). 350,000 cells per well weretransfected with 2 μg of DNA construct using 6 μl FuGENE 6 reagent(Roche, catalog number: 11-814-443-001) diluted into 94 ul DMEM. Themixture was incubated at room temperature for 15 minutes. The complexmixture was added dropwise to the cells and swirled. Cells were placedin incubator maintained at 37° C. with 5% CO2 content. 48 hoursfollowing transfection, transfected cells were transferred to a 75 cm2tissue culture flask containing 15 ml of selection media: complete mediasupplemented with 5 μg\ml puromycin (Sigma, catalog number P8833). Cellswere placed in incubator, and media was changed every 3-4 days, untilclone formation observed.

Upon sufficient quantities of cells passing through selection, 3-5million cells were harvested. Cells were lysed in 300 μl RIPA buffer (50mM Tris HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium Deoxycholate, 0.1%SDS) supplemented with protease inhibitors (Roche, catalog number:11873580001), for 20 min at 4° C. Following centrifugation at 4° C. for10 minutes at 14,000×rpm, the clear supernatants were transferred toclean tubes, and were used for WB procedure: 30 ug of lysate was mixedwith DTT 1,4-Dithiothreitol (DTT; a reducing agent) to a finalconcentration of 100 mM.

In addition, the samples were then incubated at 100° C. for 10 minutes,followed by a 1 minute spin at 14,000×rpm. SDS-PAGE (Laemmli, U.K.,Nature 1970; 227; 680-685) was performed upon loading of 30 μl of sampleper lane into a 4-12% NuPAGE® Bis-Tris gels (Invitrogen, catalog number:NP0321), and gels were run in 1×MES SDS running buffer (Invitrogen,catalog number: NP0060), using the XCell SureLock™ Mini-Cell(Invitrogen, catalog number: E10001), according to manufacturer'sinstructions. The separated proteins were transferred to anitrocellulose membrane (Schleicher & Schuell, catalog number: 401385)using the XCell™ II blotting apparatus (Invitrogen, catalog numberE19051), according to manufacturer's instructions.

The membrane containing blotted proteins was processed for antibodydetection as follows:

Non-specific regions of the membrane were blocked by incubation in 5%skim-milk diluted in Phosphate buffered saline (PBS) supplemented with0.05% Tween-20 (PBST) for 1 hour at room temperature (all subsequentincubations occur for 1 hour at room temperature). Blocking solution wasthen replaced with primary Rabbit Anti TMEM25 antibody (Cat no.HPA012163, Sigma) diluted 1:500 in 5% bovine serum albumin (BSA) (Sigma,catalog number: A4503) (diluted in PBS). After 1 hour incubation, Three5 minute washes, secondary antibody was applied: goat anti-rabbitconjugated to Peroxidase conjugated Affipure Goat anti Rabbit IgG(Jackson, catalog number: 111-035-003) diluted 1:20,000 in blockingsolution. Proteins were also detected by Mouse anti Flag M2-Peroxidase(Sigma, catalog number: A8592) diluted 1:1000 in blocking solution.After 1 hour incubation, 3×5 minute washes, ECL substrate (PIERCE,catalog number: PIR-34080) was applied for 1 minute, followed byexposure to X-ray film (Fuji, catalog number: 100NIF). The results arepresented in FIG. 23.

FIG. 23A demonstrate that Rabbit anti TMEM25 described above recognizedspecifically TMEM25_P5 protein (SEQ ID NO: 7) and TMEM25_P5_Flag (SEQ IDNO: 129) at the expected band size˜40.2 kDa, but not HEK_293 T_pRp3.

FIG. 23B demonstrate that TMEM25_P5_Flag proteins (SEQ ID NO: 129) werespecifically recognized by anti-Flag at the expected band size ˜40.2kDa.

Example 19

Determination of the Subcellular Localization of the Ectopic TMEM25_P5and TMEM25_P5_Flag in HEK293T Cells by Immunofluorescence

Protein localization of TMEM25_P5 (SEQ ID NO: 7) and of TMEM25_P5_FLAG(SEQ ID NO: 129) were observed upon Stable transfection as describedabove using confocal microscopy.

Stably transfected recombinant HEK293T cells expressing TMEM25_P5 (SEQID NO: 7) and TMEM25_P5_FLAG (SEQ ID NO: 129) were plated on coverslipspre-coated with Poly-L-Lysine (Sigma; Catalogue no. P4832). After 24 hrsthe cells were processed for immunostaining and analyzed by confocalmicroscopy.

The cover slip was washed in phosphate buffered saline (PBS), then fixedfor 15 minutes with a solution of 3.7% paraformaldehyde (PFA) (Sigma,catalog number: P-6148)13% glucose (Sigma, catalog number: G5767)(diluted in PBS). Quenching of PFA was done by a 5 minute incubation in3 mM glycine (Sigma, catalog number: G7126) (diluted in PBS). After two5-minute washes in PBS, blocking of non-specific regions was done with5% bovine serum albumin (BSA) (Sigma, catalog number: A4503) (diluted inPBS) for 20 minutes.

The coverslip was then incubated, in a humid chamber for 1 hour, withmouse anti FLAG-Cy3 antibodies (Sigma, catalog number: A9594), diluted1:200 in 5% BSA in PBS, OR with Rabbit Anti TMEM25 (Cat no. HPA012163,Sigma), diluted 1:50 in 5% BSA in PBS followed by three 5-minute washesin PBS. For the anti TMEM25 Ab only, a secondary Ab was needed: Donkeyanti Rabbit cy3 (cat#711-165-152, Jackson) diluted 1:200 in 5% BSA inPBS, incubated in a humid chamber for 1 hour, followed by three 5-minutewashes in PBS. After a prewash with BISBENZIMIDE H 33258 (HBSS) (Sigma,catalog number: 14530), the coverslip was incubated with WGA-Alexa 488(Invitrogen, catalog number W11261) diluted 1:200 in HBSS for 10 min,followed by two washes in HBSS and incubated in BISBENZIMIDE H 33258(Sigma, catalog number: 14530) diluted 1:1000 in HBSS. The coverslip wasthen mounted on a slide with Gel Mount Aqueous medium (Sigma, catalognumber: G0918) and cells were observed for the presence of fluorescentproduct using confocal microscopy.

The subcellular localization of TMEM25_P5 (SEQ ID NO:132) andTMEM25_P5_Flag (SEQ ID NO: 129) using anti TMEM25 Abs, is demonstratedin FIGS. 24A and 24B respectively. FIG. 24C demonstrates TMEM25_P5_Flag(SEQ ID NO: 129) localization using anti-FLAG Abs (Sigma, catalognumber: A9594). TMEM25_P5 protein is localized to the cell surface.

Example 20

Determining Cell Localization of TMEM25_(—) P5_Flag by FACs

Membrane localization of TMEM25_P5_Flag protein (SEQ ID NO: 129) wasobserved upon stable transfection described above, by Flow cytometryanalysis, using anti TMEM25 antibodies (Ab1628, Yomics) and by Normalmouse serum as negative control (015-000-120, Jackson). RecombinantHEK293T cells expressing TMEM25_P5_Flag were stained with anti TMEM25antibodies (A) or by Normal mouse serum (B) followed by Donkey AntiMouse-DyLight 549 conjugated secondary Ab (Jackson 715-506-150), andwere observed for the presence of fluorescent signal.

Recombinant HEK293T-TMEM25_P5_Flag cells were dissociated from the plateusing Cell dissociation buffer Enzyme-Free PBS-Based (Gibco; 13151-014),washed in FACS buffer [Dulbecco's Phosphate Buffered Saline (PBS)(Biological Industries, 02*023-1A)/1% Bovine Albumin (Sigma, A7030)] andcounted. 0.5×10̂6 cells were re-suspended in 100 μl of antibody solution,at a dilution 1:2250 ul, and incubated for 1 hour on ice. The cells werewashed with ice-cold FACS buffer and incubated with secondary antibodyas indicated for 1 hour on ice. The cells were washed with ice-cold FACSbuffer and re-suspended in 500 μl FACS buffer, then analyzed on the FACSmachine (FACSCalibur, BD). The data was acquired and analyzed usingCellquest Pro VER. 5.2.

The results presented in FIG. 25 demonstrate that anti TMEM25 antibodies(A) bind to the full length TMEM25 protein, in HEK293T recombinant cellsexpressing TMEM25_P5_Flag protein, as compare to mouse serum (B) used asa negative control, indicating membrane localization of TMEM25 protein.

Example 21 Analysis of the Expression of Endogenous TMEM25 Protein inVarious Cell Lines

The expression of endogenous TMEM25 protein in various cell lines wasanalyzed by Western Blotting as described below.

JURKAT (ATCC no. TIB-152), Daudi (ATCC no. CCL-213), RPMI8226 (ATCC no.CCL-155), G-361 (ATCC no. CRL-1424), KARPAS (ATCC no. VR-702) cellextracts were prepared as described above (Lanes 3-7 in FIG. 26—seefigure legend for the corresponding lane/material assignments).

Whole cell lysates were prepared and analyzed by western blot asdescribed above. Equal protein amounts were analyzed by SDS-PAGE andtransferred to Nitrocellulose membrane as described above.

The membrane was blocked by 5% skim-milk diluted in Phosphate bufferedsaline (PBS) supplemented with 0.05% Tween-20 (PBST) for 1 hourincubation at room temperature (all subsequent incubations occur for 1hour at room temperature). Blocking solution was then replaced withprimary Rabbit Anti TMEM25 antibody (Cat no. HPA012163, Sigma) diluted1:500 in 5% bovine serum albumin (BSA) (Sigma, catalog number: A4503)(diluted in PBS). After 1 hour incubation, Three 5 minute washes,secondary antibody was applied: goat anti-rabbit conjugated toPeroxidase conjugated Affipure Goat anti Rabbit IgG (Jackson, catalognumber: 111-035-003) diluted 1:20,000 in blocking solution. Proteinswere also detected by Mouse anti Flag M2-Peroxidase (Sigma, catalognumber: A8592) diluted 1:1000 in blocking solution. After 1 hourincubation, 3×5 minute washes, ECL substrate (PIERCE, catalog number:PIR-34080) was applied for 1 minute, followed by exposure to X-ray film(Fuji, catalog number: 100NIF).

FIG. 26 demonstrates the endogenous expression of TMEM25 in various celllines. A protein at 40.2 kDa corresponding to TMEM25 as observed inHEK293T cells expressing TMEM25_P5_Flag (lane 2; lane 1 shows a controlwithout Flag), detected with anti TMEM25 antibody in extracts ofRPMI8226 (lane 5), Daudi (lane 6) and JURKAT (lane 7).

Example 22

Transfection of Stable HEK293T TMEM25 with siRNA to TMEM25

Specific knockdown of TMEM25_P5_Flag protein (SEQ ID NO:129) expressionwas observed in HEK293T cells stably expressing TMEM25_P5_Flag (SEQ IDNO 129) previously described upon transfection with TMEM25_P5-SiRNAs.

siRNA was purchased from Dharmacon as follows: TMEM25 (L-018183-00-0005,Dharmacon, ON TARGET plus SMART pool, Human TMEM25 (84866), 5nmol) andscrambled SiRNA as a negative control (Dharmacon, D-001810-10-05).

Cells were plated at 50-70% confluence 24 hr prior to transfection.siRNA complexes at 250pmol were added to 250 ul reduced serum Opti-MEM(cat 31985, GIBCO). In parallel, Lipofectamine 2000 reagent(cat#11668019, Invitrogen) was mixed; 5 ul was added to 250 ul reducedserum Opti-MEM (cat 31985, GIBCO). Tubes were combined and incubated for15-30 min at RT for sufficient complexes to form; the material was thendistributed over the cells and incubated for 48 hr. Cells were harvestedand cell lysates prepared as described above and detected by anti TMEM25(Cat no. HPA012163, Sigma), following by secondary Donkey anti Rabbitconjugated to Peroxidase.

FIG. 27 demonstrates specific knockdown of TMEM25_P5_Flag protein (SEQID NO: 129) in HEK293T cells stably expressing TMEM25_P5_Flag (SEQ ID NO129) transfected with TMEM25_P5 siRNA (L-018183-00-0005, Dharmacon)(Lane 2) compared to HEK293T cells stably expressing TMEM25_P5_FLAGtransfected with Scrambled-SiRNA (Lane 1) (Dharmacon, D-001810-10-05),using anti TMEM25 antibodies (Sigma, cat# HPA012163).

Example 23 Immunohistochemistry (IHC) Using Anti LSR and Anti TMEM25Poly Clonal Antibodies

To assess the tissue binding profiles, anti-LSR (Abcam catalog number:ab59646) and Anti TMEM25 (Cat no. HPA012163, Sigma), were applied on apanel of tumor tissues microarray (TMA), as detailed in Table 10.

HEK-293 cells expressing LSR_P5a_Flag_m (SEQ ID NO 144) orTMEM25_P5_Flag (SEQ ID NO:129) were used as a positive control forcalibration of the pAb for staining.HEK293T cells transfected with emptyvector were used as a negative controls as well as rabbit serum IgGantibodies.

The immunohistochemical detection of LSR_P5a_Flag_m (SEQ ID NO:144) orwith TMEM25_P5_Flag (SEQ ID NO:129) by the antibodies anti-LSR and AntiTMEM25 accordingly, were calibrated in formalin-Fixed paraffin-embedded(FFPE) sections. Two antigen retrieval methods were used: pH6.1 andpH9.0 in three Abs concentrations (3, 1, 0.3 ug/ml).

The antigen retrieval methods were performed as follows. The abovedescribed FFPE sections were deparaffinized, antigen retrieved andrehydrated using pH6.1 or pH9.0 Flex+ 3-in-1 antigen retrieval buffersand a PT Link automated antigen retrieval system, at 95° C. for 20 minwith automatic heating and cooling.

Following antigen retrieval, sections were washed in distilled water for2×5 min then loaded into a DAKO Autostainer Plus. The sections were thenincubated for 10 min with Flex+ Peroxidase Blocking reagent, rinsedtwice in 50 mM Tris.HCl, 300 mM NaCl, 0.1% Tween-20, pH 7.6 (TBST),followed by a 10 min incubation with Protein Block reagent (DAKO X0909).

The sections were incubated for 30 min with primary antibody diluted inDAKO Envision Flex antibody diluent (DAKO Cytomation, Cat # K8006).Following incubation with primary antibodies, the sections were thenrinsed twice in FLEX buffer, incubated with anti-mouse/rabbit Flex+ HRPfor 20 min, rinsed twice in FLEX buffer and then incubated withdiaminobenzidine (DAB) substrate for 10 min. The chromagenic reactionwas stopped by rinsing the slides with distilled water.

Following chromagenesis, the sections were counterstained withhaematoxylin, dehydrated in an ascending series of ethanols(90-99-100%), cleared in three changes of xylene and coverslipped underDePeX. Stained sections were analysed by using an Olympus BX51microscope with a Leica DFC290 camera.

FIG. 28 demonstrates that anti LSR antibody (Cat no. ab59646, Abcam) insections of positive control cell line (panels A, C and E) showedspecific immunoreactivity in a dose dependent concentrations of 3.1 and0.3 ug/ml respectively, as compared to the negative control cell line(panels B, D and F), in pH 9, according to the antigen retrieval methodpreviously described.

FIG. 29 demonstrates that anti TMEM25 (Cat no. HPA012163, Sigma) insections of positive control cell line (panels A, C and E) showsspecific immunoreactivity in a dose dependent concentrations of 3.1 and0.3 ug/ml respectively, as compared to the negative control cell line(panels B, D and F), in pH 9, according to the antigen retrieval methodpreviously described.

TABLE 10 Summary of the tissue samples included in the tissue microarray(TMA). TMA Map (X, Y) Donor ID position ID Tissue Path report Age Sex 1(1, 1) 15766 tumour:breast:lobular carcinoma Infiltrating lobularcarcinoma. 42 Female Grade2/3 2 (2, 1) 5252 tumour:breast:ductal- Thisslide contains a sample of an in 57 Female adenocarcinoma situ andinfiltrating ductal carcinoma (modified Bloom and Richardson grade III).Breast - in situ and infiltrating ductal carcinoma. 3 (3, 1) 8723tumour:breast:ductal- Primary breast cancer (invasive 74 Femaleadenocarcinoma ductal pattern) 4 (4, 1) 15778 tumour:breast:lobularcarcinoma Sections of skin with dermis and 52 Female subcutisinfiltrated by poorly differentiated, slightly discohesive carcinoma.Individual cells have rather pleomorphic nuclei. Appearances areconsistent with a pleomorphic lobular carcinoma. 5 (5, 1) 3724tumour:breast:ductal- Invasive and in situ ductal carcinoma 82 Femaleadenocarcinoma of breast. 6 (6, 1) 2953 tumour:breast:ductal- Thespecimen consists of breast 67 Female adenocarcinoma tissue includingDCIS (ductal carcinoma in situ) and widespread invasive poorlydifferentiated adenocarcinoma. 7 (7, 1) 9533 tumour:breast:ductal- Thisslide contains breast tissues 50 Female adenocarcinoma infiltrated by apoorly differentiated ductal carcinoma. Breast tumour - ductalcarcinoma. 8 (8, 1) 3346 tumour:breast:ductal- The specimen consists of63 Female adenocarcinoma connective tissue elements widely infiltratedby a poorly differentiated ductal adenocarcinoma. 9 (9, 1) 5704 breastThis section contains a good sample 46 Female of normal breast tissue 10(10, 1)  5347 breast Normal breast 64 Female 11 (11, 1)  3550tumour:colon:adenocarcinoma The large bowel is widely 61 Maleinfiltrated by a moderately well differentiated adenocarcinomaconsistent with a derivation from the colon. 12 (12, 1)  3269tumour:colon:adenocarcinoma Primary colonic pattern 58 Maleadenocarcinoma (moderately differentiated). 13 (1, 2) 15767tumour:colon:adenocarcinoma Moderately differentiated 58 Femaleadenocarcinoma. 14 (2, 2) 3751 tumour:colon:adenocarcinoma Moderatelydifferentiated 79 Female adenocarcinoma. 15 (3, 2) 3881tumour:colon:adenocarcinoma Moderately differentiated 71 Maleadenocarcinoma. 16 (4, 2) 2889 tumour:colon:adenocarcinoma The specimenconsists of large 73 Female bowel showing surface ulceration associatedwith a moderately well differentiated primary adenocarcinoma. 17 (5, 2)15764 tumour:colon:adenocarcinoma Moderately differentiated 75 Femaleadenocarcinoma. 18 (6, 2) 15763 tumour:colon:adenocarcinoma Moderatelydifferentiated 69 Female adenocarcinoma. 19 (7, 2) 2681 colon Normalcolon: full thickness. 54 Female 20 (8, 2) 3121 colon Full thicknessnormal colon. Colon - 34 Male normal. 21 (9, 2) 5638 tumour:prostateProstate tumour - adenocarcinoma 87 Male consistent with an origin inprostate. Gleason score 5 + 5 = 10. 22 (10, 2)  15295tumour:prostate:adenocarcinoma Adenocarcinoma. Gleason Score 71 Male 3 +3 = 6 23 (11, 2)  15301 tumour:prostate:adenocarcinoma Adenocarcinoma.Gleason Score 51 Male 3 + 4 = 7 24 (12, 2)  15758tumour:prostate:adenocarcinoma Adenocarcinoma. Gleason Score 74 Male 3 +4 = 7 25 (1, 3) 15745 tumour:prostate:adenocarcinoma Adenocarcinoma.Gleason Score 52 Male 4 + 5 = 9 26 (2, 3) 15777tumour:prostate:adenocarcinoma Adenocarcinoma. Gleason Score 68 Male 4 +4 = 8 27 (3, 3) 15755 tumour:prostate:adenocarcinoma Adenocarcinoma.Gleason Score 55 Male 3 + 4 = 7 28 (4, 3) 15756tumour:prostate:adenocarcinoma Adenocarcinoma. Gleason Score 68 Male 4 +5 = 9 29 (5, 3) 1317 prostate Normal prostate 55 Male 30 (6, 3) 13951prostate Normal prostate 37 Male 31 (7, 3) 15052 Lymphoma Lymph nodeinfiltrated by large cell 45 Female lymphoma 32 (8, 3) 15760 LymphomaLow Grade Non-Hodgkin's 72 Female Lymphoma 33 (9, 3) 15754 Lymphoma HighGrade Non-Hodgkin's 77 Male Lymphoma 34 (10, 3)  15039 LymphomaInfiltrate of medium to large size 47 Male lymphocytes with high mitoticrates. High grade Non-Hodgkin's Lymphoma. 35 (11, 3)  15034 LymphomaDiffuse infiltrate of monotamous 71 Male lymphoid cells consistent withNon-Hodgkin's Lymphoma. 36 (12, 3)  15037 Lymphoma Diffuse infiltrate ofmonotamous 53 Female lymphoid cells consistent with Non-Hodgkin'sLymphoma. Thyroid tissue seen on edge of section. 37 (1, 4) 15032Lymphoma Diffuse infiltrate of small 50 Female lymphocytes consistentwith Non-Hodgkin's Lymphoma. 38 (2, 4) 15775 Lymphoma Hodgkin's Lymphoma75 Female 39 (3, 4) 4655 lymph-node Lymph node within normal limits. 1Female 40 (4, 4) 10789 lymph-node Normal lymph node. 58 Male 41 (5, 4)12053 tumour:lung Poorly differentiated non- 72 Male small cellcarcinoma with some squamoid features. NON SMALL CELL CARCINOMA 42 (6,4) 15772 tumour: lung: non-small cell Poorly differentiated non-smallcell 44 Male carcinoma carcinoma 43 (7, 4) 13586 tumour:lung Moderatelyto poorly differentiated 67 Female squamous carcinoma. 44 (8, 4) 2760tumour:lung:squamous-cell- The specimen includes normal 64 Malecarcinoma bronchus, a large vessel presumed to be an artery showingextensive intimal fibrosis/organisation as well as lung parenchymawidely infiltrated by a moderately well differentiated keratinisingsquamous cell carcinoma. 45 (9, 4) 9354 tumour:lung:adenocarcinomaSection of lung tissue containing 63 Male a tumour growing along thealveolar spaces. The tumour is of large cell type showing features of anadenocarcinoma. 46 (10, 4)  3473 tumour:lung:adenocarcinoma Lungtumour - poorly 72 Male differentiated adenocarcinoma consistent with aprimary origin in lung if an origin elsewhere can be excluded. 47 (11,4)  5757 tumour:lung:adenocarcinoma Lung tumour - adenocarcinoma of 72Male broncho-alveolar pattern. 48 (12, 4)  4852tumour:lung:adenocarcinoma Lung tumour - adenocarcinoma 56 Female withprominent broncho-alveolar pattern. 49 (1, 5) 10414 small cell Sectionsof lung showing a 74 Male poorly differentiated, small cell carcinoma.DIAGNOSIS: Lung; small cell carcinoma. 50 (2, 5) 15055 small cellFibrous tissue infiltrated by small 52 Male cell carcinoma 51 (3, 5)15054 small cell Sections of lung infiltrated by small 65 Male cellcarcinoma 52 (4, 5) 15053 small cell Sections of lung infiltrated bysmall 52 Male cell carcinoma 53 (5, 5) 1311 lung:parenchyma Lung withinnormal limits. 36 Female 54 (6, 5) 14 lung:parenchyma Normal lung andbronchus. 39 Female 55 (7, 5) 5767 lung:parenchyma Lung parenchyma(including pleural 45 Male surface) - normal limits. 56 (8, 5) 2649lung:parenchyma Normal lung 37 Male 57 (9, 5) 3588 tumour:stomach Biopsyshows poorly differentiated 69 Female mucinous carcinoma. 58 (10, 5) 5065 tumour:stomach Sections show a well 64 Male differentiatedadenocarcinoma of the stomach. 59 (11, 5)  9275 tumour:stomach Sectionsof stomach antrum 78 Female showing a moderately differentiated,infiltrating adenocarcinoma. The carcinoma is seen in both the mucosaand infiltrating the submucosa. DIAGNOSIS: gastric carcinoma. 60 (12,5)  2295 stomach Section shows a moderately 66 Female differentiatedadenocarcinoma of the stomach. 61 (1, 6) 13665 stomach:body Fullthickness section of normal 57 Female stomach compatible with body. 62(2, 6) 2874 stomach:body Stomach - full thickness wall with 53 Malenormal body type mucosa. 63 (3, 6) 12998 tumour:ovary A serous papillarycystic carcinoma. 78 Female 64 (4, 6) 13003 tumour:ovary Invasive serouspapillary carcinoma. 74 Female 65 (5, 6) 5739 tumour:ovary Sections ofovary showing 48 Female infiltrating islands of cohesive cells in whichthere are nuclei showing nuclear grooving. The appearances areconsistent with a granulosa cell tumour. ovary; granulosa cell tumour.66 (6, 6) 9407 tumour:ovary This slide contains a portion from 75 Femalethe wall of a multi loculated ovarian tumour with a pattern bestclassified as serous cystadenocarcinoma. Ovary tumour - serouscystadenocarcinoma. 67 (7, 6) 4739 ovary This is normal ovarian tissue42 Female showing follicular structures (primordial follicles and acystic follicle) and an involuting corpus luteum. 68 (8, 6) 4781 ovaryNormal ovarian cortex with follicles. 34 Female 69 (9, 6) 15759 melanomaMalignant melanoma 65 Male 70 (10, 6)  15753 melanoma High grademalignant melanoma 46 Female 71 (11, 6)  15038 melanoma Sections of skinwith ulcerated 41 Male surface with a large dermal deposit of malignantmelanoma 72 (12, 6)  15343 melanoma Malignant melanoma 24 Male 73 (1, 7)13779 skin This slide contains a well 44 Female orientated section ofnormal skin including some subcutis. Hair follicles are few in number,sebaceous glands are few and sweat glands are moderately abundant. Skin,breast - normal. 74 (2, 7) 13280 skin Normal skin including dermis and50 Female epidermis. 75 (3, 7) 15342 tumour:brain:glioblastoma Sectionsof brain of a very cellular 56 Male multiforme tumour composed of glialcells demonstrating nuclear pleiomorphism and focal necrosis 76 (4, 7)9514 tumour:brain Sections shows brain tissue 17 Male infiltrated by anAstrocytoma; grade 2. 77 (5, 7) 3306 tumour:brain Sections show aspindle cell 82 Male meningioma. 78 (6, 7) 9516 tumour:brain Sectionsshows brain tissue 25 Female infiltrated by an Astrocytoma; grade 4. 79(7, 7) 2007 brain:cortex:frontal Normal brain 40 Male 80 (8, 7) 4585brain:cortex:frontal Sections show normal grey matter 85 Male of thecortex containing unremarkable neurones and this overlies normal whitematter. normal brain cortex. 81 (9, 7) 3737 tumour:kidney The specimenshows the 54 Female features of a primary renal cell adenocarcinoma. 82(10, 7)  13262 tumour:kidney Grade 1 papillary transitional cell 59 Malecarcinoma 83 (11, 7)  4764 tumour:kidney Renal cell (clear cell)carcinoma 66 Male 84 (12, 7)  9043 tumour:kidney Clear cell renal cellcarcinoma of 45 Male kidney. 85 (1, 8) 2874 kidney:cortex Normal renalcortex 53 Male 86 (2, 8) 4818 kidney:cortex Normal renal cortex. 52Female 87 (3, 8) 14022 tumour:liver Poorly differentiated 45 Malecholangiocarcinoma 88 (4, 8) 15757 tumour:liver Fibrolamellarhepatocellular 25 Male carcinoma 89 (5, 8) 14826 tumour:liver Low Gradehepatocellular carcinoma 66 Female 90 (6, 8) 15750 tumour:liverCholangiocarcinoma 70 Female 91 (7, 8) 1991 liver:parenchyma Normalliver 79 Female 92 (8, 8) 3123 liver:parenchyma Liver - normal limits.31 Male

Example 24 Full Length Validation of Encoding LY6G6F Transcript

A full Length transcript encoding LY6G6F (SEQ ID NO: 1) was validated asdescribed below:

1. A reverse transcription reaction was carried out as follows: 10 μg ofpurified RNA (lung normal) was mixed with 150 ng Random Hexamer primers(Invitrogen, Carlsbad, Calif., USA, catalog number: 48190-011) and 500μM dNTPs in a total volume of 156 μl . The mixture was incubated for 5mM at 65° C. and then quickly chilled on ice. Thereafter, 50 μl of 5×SuperscriptII first strand buffer (Invitrogen, catalog number:18064-014, part number: Y00146), 24 μl 0.1M DTT and 400 units RNasin(Promega, Milwaukee, Wis., U.S.A., catalog number: N2511) were added,and the mixture was incubated for 10 mM at 25° C., followed by furtherincubation at 42° C. for 2 mM. Then, 10 μl (2000 units) of SuperscriptII(Invitrogen, catalog number: 18064-014) was added and the reaction(final volume of 2500 was incubated for 50 mM at 42° C. and theninactivated at 70° C. for 15 min. The resulting cDNA was diluted 1:20 inTE buffer (10 mM Tris, 1 mM EDTA pH 8).2. PCR was done using 2× GoTaq ReadyMix (Promega, catalog number:M7122.) under the following conditions: 12.5 ul GoTaq ready mix; 5 ulcDNA from the above; 1 ul of 10 uM forward primer 100-690 (SEQ IDNO:51); 1 ul of 10 uM reverse primer 100-691 (SEQ ID NO:52) and 5.5 ulH₂O in a total reaction volume of 25 μl ; with a reaction program of 5minutes in 95° C.; 35 cycles of: 30 seconds at 94° C., 30 seconds at 53°C., 50 seconds at 72° C.; then 10 minutes at 72° C. The detailsregarding the primers are presented in Table 11 below.

The PCR product above was loaded on 1.2% agarose gel stained withethidium bromide, electrophoresed in 1×TAE solution at 100V, andvisualized with UV light. The expected band size was excised andextracted from the gel using QiaQuick™ Gel Extraction kit (Qiagen,catalog number: 28707). The purified DNA was then sequenced (Tel-AvivUniversity, Israel) using the above primers and was verified for thefull length LY6G6F encoding transcript (SEQ ID NO:1).

Example 25 Cloning of Full Length Transcript Encoding LY6G6F Fused toEgypt

Cloning of Full Length transcript encoding LY6G6F fused to EGFP(Enhanced Green Fluorescent Protein) was performed as described below.

First, an EGFP expression vector was constructed and then the LY6G6Fopen reading frame (SEQ ID NO:57), encoding the amino acid sequence setforth in SEQ ID NO:58, was cloned. EGFP was subcloned into pIRESpuro3(Clontech catalog number: 631619) as follows: EGFP-N1 vector (Clontechcatalogue number: 6085-1) was digested with NheI and NotI to excise theEGFP gene. The EGFP insert was then ligated into pIRESpuro3 (Clontechcataloge number: 631619), which was previously digested with the sameenzymes, in order to obtain the EGFP-pIRESpuro3 vector.

PCR was done using Platinum PFX™ (Invitrogen, Carlsbad, Calif., USA,catalog number: 1178-021) under the following conditions: 5 μl PlatinumPFX 10× buffer; 2 μl—purified validated DNA from the above; 1 μl-10 mMdNTPs (2.5 mM of each nucleotide); 1 μl—Platinum PFX enzyme; 37 μl-H2O;1 μl of 10 uM forward primer 100-729 (SEQ ID NO:53); 1 ul of 10 uMreverse primer 100-730 (SEQ ID NO:54) (10 μM each) in a total reactionvolume of 50 μl ; with a reaction program of 5 minutes in 95° C.; 35cycles of: 30 seconds at 94° C., 30 seconds at 55° C., 60 seconds at 68°C.; then 10 minutes at 68° C. Primers which were used included genespecific sequences corresponding to the desired coordinates of theprotein and restriction enzyme sites and Kozak sequence, as listed inTable 11, below and in FIG. 6. Bold letters in Table 11 represent thespecific gene sequence while the restriction site extensions utilizedfor cloning purposes are in Italic and Kozak sequence are underlined.

5 μl of the PCR product above, were loaded on 1.2% agarose gel stainedwith ethidium bromide, electrophoresed in 1×TAE solution at 100V, andvisualized with UV light. After verification of expected size band,remaining PCR product was processed for DNA purification using QiaquickPCR purification kit (Qiagen™, Valencia, Calif., U.S.A., catalog number28106). The extracted PCR product were digested with NheI and EcoRIrestriction enzymes (New England Biolabs, Beverly, Mass., U.S.A.), aslisted in Table 11. After digestion, DNAs were loaded onto a 1.2%agarose gel as described above. The expected band size was excised andextracted from the gel using QiaQuick™ Gel Extraction kit (Qiagen,catalog number: 28707).

The digested DNA was ligated to EGFP_pIRESpuro3 vector previouslydigested with NheI and EcoRI restriction enzymes, using the LigaFast™Rapid DNA Ligation System (Promega, catalog number: M8221). Theresulting DNA was transformed into competent E. Coli bacteria DH5a (RBCBioscience, Taipei, Taiwan, catalog number: RH816) according tomanufacturer's instructions, then plated on LB-ampicillin agar platesfor selection of recombinant plasmids, and incubated overnight at 37° C.

Screening positive clones was performed by PCR using GoTaq Ready Mix(Promega, catalog number: M7122). Positive colonies were grown in 5 mlTerrific Broth supplemented with 100 μg/ml ampicillin, with shakingovernight at 37° C. Plasmid DNA was isolated from bacterial culturesusing Qiaprep™ Spin Miniprep Kit (Qiagen, catalog number: 27106).Accurate cloning was verified by sequencing the inserts (Tel AvivUniversity, Israel). Upon verification of an error-free colony (i.e. nomutations within the ORF), recombinant plasmids were processed forfurther analysis.

The DNA sequence of the resulting LY6G6F full length_fused to EGFP (SEQID NO:55) is shown in FIG. 7. In FIG. 7 gene specific sequencecorresponding to the LY6G6F full length sequence is marked in bold facedtype, while the EGFP sequence is marked in Italics and underlining. Theamino acid sequence of the resulting LY6G6F full length fused to EGFP(SEQ ID NO:56) is shown in FIG. 8; gene specific sequence correspondingto the full length sequence of LY6G6F is marked in bold faced type,while the EGFP sequence is marked in Italics and underlining.

TABLE 11 primer details SEQ Primer Restriction ID NO: ID Primer sequencesite 51 100-690 GAGAACTTGGCAGGCTCTCC — 52 100-691 CACACTTCCCAGCAGATGTC —53 100-729 CTAGCTA GCCACC ATGGCAGTC NheI TTATTCCTCCTC 54 100-730CGCGAATTC GCCTGGGCTTGT EcoRI GGGCAGGTG

Example 26 Determining Cell Localization of LY6G6F

In order to determine the cellular localization of the LY6G6F protein,LY6G6F-EGFP fusion protein (SEQ ID NO:56) was used. LY6G6F proteinlocalization was observed upon transient transfection (Chen et al.,Molecular vision 2002; 8; 372-388) using the confocal microscope. Thecells were observed for the presence of fluorescent products 48 hoursfollowing transfection.

The LY6G6F-EGFP pIRESpuro3 construct, described above, was transientlytransfected into HEK-293T cells as follows:

HEK-293T (ATCC, CRL-11268) cells were plated on sterile glasscoverslips, 13 mm diameter (Marienfeld, catalog number: 01 115 30),which were placed in a 6 well plate, using 2 ml pre-warmed DMEM[Dulbecco's modified Eagle's Media, Biological Industries (Beit Ha'Emek,Israel), cataloge number: 01-055-1A]+10% FBS (Fetal Bovine Serum)+4 mML-Glutamine 500,000 cells per well were transfected with 2 μg of the DNAconstruct using 6 μl FuGENE 6 reagent (Roche, catalog number:11-814-443-001) diluted into 94 ul DMEM. The mixture was incubated atroom temperature for 15 minutes. The complex mixture was added dropwiseto the cells and swirled. Cells were placed in an incubator maintainedat 37° C. with 5% CO2 content.

48 hours post transient transfection the cells were further processedfor analysis in confocal microscopy. The cover slips were washed 3 timesin phosphate buffered saline (PBS) and fixed for 15 minutes with 3.7%paraformaldehyde (PFA) (Sigma, catalog number: P-6148). After 2 washesin PBS, the fixed coverslips were glued to a slide using mountingsolution (Sigma, catalog number: G0918) and cells were observed for thepresence of fluorescent product using confocal microscope. The resultsare presented in FIG. 9.

FIG. 9 demonstrates that the LY6G6F_EGFP (SEQ ID NO:56) fused proteinlocalizes to cell membrane upon expression in HEK 293T cells. The imagewas obtained using the 40× objective of the confocal microscope.

Example 27 Cloning and Expression of the LY6G6F, VSIG10, TMEM25 and LSRECD-Mouse IGg2A-FC Fused Proteins

Mouse orthologs of human LY6G6F, VSIG10, TMEM25, and LSR proteins wereidentified using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters and used to gainexperimental proof of concept related to the functionality of theLY6G6F, VSIG10, TMEM25 and/or LSR Ig fusion proteins in animal model.The mouse orthologs corresponding to human LY6G6F, VSIG10, TMEM25 andLSR proteins are shown in SEQ ID NOs: 20, 19, 9 and 21, respectively.The amino acid alignment and comparison of the human LY6G6F, VSIG10, LSRand TMEM25 proteins to the respective mouse orthologs is shown in FIGS.5A, 5B, 5C and 5D respectively.

cDNA sequence mouse TMEM25 (SEQ ID NO:9), LY6G6F (SEQ ID NO:20), VSIG10(SEQ ID NO:19), and LSR (SEQ ID NO: 21) proteins were each fused to theFc domain of mouse IgG2aFc (SEQ ID NO: 27). In all cases the naturalcorresponding signal peptide was used for each ECD. The resulted LY6G6F,VSIG10, TMEM25 or LSR ECD-mIgG2aFc Ig fused proteins (SEQ ID NOs: 23,24, 25, or 26, respectively) are shown in FIGS. 10A-D, respectively.

The LY6G6F, VSIG10, TMEM25 or LSR ECD-mIgG2aFc fused proteins (SEQ IDNOs: 23, 24, 25, or 26, respectively), were cloned into GPEx®retrovectors, followed by retrovector transduction into Catalent's“in-house” CHO—S cell line. A pooled population was produced and theproductivity was validated. The pool was then expanded and relativeproductivity and relative copy number of the pool was determined. Cellculture supernatants were analyzed by Catalent's Fc ELISA assay toconfirm production of LY6G6F, VSIG10, TMEM25 or LSR ECD-mIgG2aFc fusedproteins.

Protein solutions were tested for bioburden and endotoxin. Human fusionproteins composed of the human ECD of either of LY6G6F, VSIG10, TMEM25or LSR ECD fused to human IgG1 (as depicted on FIG. 11) were alsoexpressed using a similar system.

Assessment of the Effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig FusionProteins on Mouse and Human T Cell Activation In Vitro Example 28

Effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig Fusion Proteins onActivation of Do11.10 Naïve Cd4+ T Cells with Ova Peptide

Naive CD4⁺ T cells were isolated from spleens of five D011.10 mice(Jackson) via automax sort: CD4-negative sort (MiltenyiCat#130-095-248), including anti-CD25 (Miltenyi Cat#130-091-072) in thenegative sort cocktail, followed by CD62L-positive sort (Miltenyi Cat#130-049-701). Balb/c total splenocytes were also collected from onemouse, and irradiated with 3000rads to serve as antigen presenting cells(APCs) for the DO11.10 CD4⁺ T cells. Naive CD4⁺ T cells were cultured at5×10⁵ cells per well in flat-bottom 96-well plates with irradiated APCsat a ratio of 1:1 (APCs to T cells) in 200 ul of HL-1 medium, andactivated with 20 ug/ml or 2 ug/ml OVA323-339 in the presence of eitherTMEM25-ECD-Ig (SEQ ID NO:25), LSR-ECD-Ig (SEQ ID NO:26), LY6G6F-ECD-Ig(SEQ ID NO:23) at the indicated concentrations. As positive controls,B7-H4-Ig (R&D Systems) or CTA4-Ig (mouse ECD fused to mIgG2a Fc) wereused. Isotype control Ig (mIgG2a, BioXCell Cat. # BE0085) was used as anegative control. The cells were pulsed with 1uCi of tritiated-thymidineat 24 hours, and harvested at 72 hours.

As shown in FIG. 30, TMEM25-ECD-Ig, LSR-ECD-Ig and LY6G6F-ECD-Ig elicitdose dependent inhibition of T cell activation. This was demonstrated asinhibition of T cell proliferation which was induced by OVA323-339 at 20ug/ml (FIGS. 30 A-C, E) or 2 ug/ml (FIG. 30 D).

VSIG10-ECD-Ig fusion protein (SEQ ID NO:24) did not show activity inthree experiments carried out in similar assay.

Example 29

Effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig Fusion Proteins onActivation of Naïve Cd4⁺ T Cells with Anti-Cd3/Anti-Cd28 Coated Beads

Naive CD4⁺ T cells were isolated from 5 SJL (Harlan) mice via automaxsort as described in the previous section. Beads were coated withanti-CD3 (0.5 ug/ml; clone 2C11) and anti-CD28 (2 ug/ml; clone 37.51eBioscience) following manufacturer's protocol (Dynabeads M-450 EpoxyCat. 140.11, Invitrogen), and with increasing concentrations ofLSR-ECD-Ig or mIgG2a isotype control (mIgG2a, BioXCell Cat. # BE0085)(0.1-10 ug/ml). The total amount of protein used for beads coating withLSR-ECD-Ig was completed to 10 ug/ml with Control Ig. Naive CD4⁺ T cells(0.5×10⁶/well) were activated with the coated beads at a ratio of 1:2(beads to T cells). The cells were pulsed with 1uCi oftritiated-thymidine after 24 hours, and harvested after 72.

LSR-ECD-Ig (SEQ ID NO:26) pronouncedly inhibited proliferation of T cellproliferation and elicit its effect in a dose dependent manner (FIG.31).

TMEM25, LY6G6F and VSIG10 ECD Ig fusion proteins shown in FIGS. 10 and11 are tested in a similar assay with similar results.

Example 30

Dose Response Effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig FusionProteins on Mouse Cd4+ T Cell Activation with Plate Bound Anti-Cd3, asManifested in Cytokine Production and Expression of the ActivationMarker Cd69.

Untouched CD4+CD25− T cells were isolated from pools of spleen and lymphnode cells of BALB/C mice by negative selection using CD4+CD62L+ T cellisolation Kit (Miltenyi Cat#130-093-227) according to the manufacturer'sinstructions. The purity obtained was >95%.

Tissue culture 96-well plates were coated overnight at 4° C. with 2ug/ml anti-CD3 mAb (clone 145-2C11) in the presence of LY6G6F, VSIG10,TMEM25 and LSR ECD-Ig fusion proteins (SEQ ID NOs: 23, 24, 25 and 26,respectively) at 1, 5 and 10 μg/ml. Control mIgG2a (Clone C1.18.4 fromBioXCell; Cat#BE0085) was added to each well in order to complete atotal protein concentration of 12 μg/ml per well. Wells were plated with1×10⁵ CD4+CD25− T cells per well. At 48 hrs post stimulation, culturesupernatants were collected and analyzed using mouse IFNγ ELISA kit, andcells were analyzed for expression of the activation marker CD69 by flowcytometry.

The results shown in FIG. 32 demonstrate inhibitory effects of LY6G6F,VSIG10, TMEM25 and LSR ECD-Ig fusion proteins on CD4 T cell activation,manifested by reduced IFNγ secretion (FIG. 32A) and reduced expressionof CD69 (FIG. 324B) upon TCR stimulation, compared to control mIgG2a andCTLA4-Ig.

Example 31 The Effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig FusionProteins on Cd4+ T Cell Differentiation In Vitro.

To test the ability of LY6G6F, VSIG10, TMEM25 and LSR Ig fusion proteinsto inhibit CD4+ T cell differentiation, naïve CD4+ T cells are isolatedfrom D011.10 mice, which are transgenic for a T cell receptor (TCR) thatis specific for OVA323-339 peptide. Using D011.10 T cells enablesstudying both polyclonal (anti-CD3/anti-CD28 mAbs) and peptide-specificresponses on the same population of CD4+ T cells. Naïve CD4+ T cells areisolated from D011.10 mice and activated in culture in the presence ofanti-CD3/anti-CD28 coated beads or OVA323-339 peptide plus irradiatedBALB/c splenocytes, in the presence of LY6G6F, VSIG10, TMEM25 or LSRECD-Ig fusion proteins, control Ig, or B7-H4 Ig. The cells are activatedin the presence of Th driving conditions as follows: Th0 cell- (IL-2),Th1 cell- (IL-2+IL-12), Th2 cell- (IL-2+IL-4), or Th17 cell-(TGF-β+IL-6+IL-23+anti-IL-2). The effects on T cell differentiation andTh-specific responses are assessed by measuring cell proliferation andsubtype specific cytokine production: IL-4, IL-5, IL-10, IL-17, IFN-γ.

Example 32 Assessment of the Effect of LY6G6F, VSIG10, TMEM25 or LSR ECDIg Fusion Proteins on Human T Cells Activation.

The effect of LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins onhuman T cell response is tested by two different in vitro assays usingpurified human T cells. In the first assay, human T cells are activatedby anti-CD3 and anti-CD28 coated beads, and in the other assay,activation is carried out using anti-CD3 and anti-CD28 antibodies in thepresence of autologous, irradiated PBMCs. The regulatory activity ofLY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins on human T cellactivation, is evaluated by measuring cell proliferation and cytokinerelease.

Study I— Activation of Human T Cells with Anti-CD3 and Anti-CD28-CoatedBeads is Inhibited by Fusion Proteins

Naïve CD4+ T cells are isolated from 4 healthy human donors andactivated with anti-CD3 mAb/anti-CD28 mAb coated beads in the presenceof control mIgG2a, or any one of the LY6G6F, VSIG10, TMEM25 or LSR ECDIg fusion proteins. Two side-by-side culture sets are set up; oneculture being pulsed at 24 hours with tritiated-thymidine and harvestedat 72 hours while the second plate is harvested at 96 hours for cytokineproduction via LiquiChip.

Study II— Activation of Human T Cells with Irradiated Autologous PBMCsis Inhibited by Fusion Proteins

Total PBMCs are isolated from fresh blood of healthy human donors usingficoll gradient. 10×10⁶ total PBMCs are resuspended in Ex-Vivo 20medium, and irradiated at 3000rad. These cells are used to activate theisolated T cells in vitro, by presenting the anti-CD3, anti-CD28 andeither of the test proteins. The rest of PBMCs are used for isolation ofT cells using CD4+ T cell Isolation Kit II from Miltenyi.

For activation, 5×10⁵ isolated T cells are cultured in the presence of5×10⁵ autologous irradiate PBMCs. Anti-CD3 (0.5 μg/ml), anti-CD28 (2μg/ml) and either of LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusionproteins or control Ig (mIgG2a) are added in a soluble form. Thecultures are pulsed with 1uCi of triated thymidine at 24 hrs, andproliferation is measured at 72 hours.

Example 33 The Effect of LY6G6F, TMEM25 and LSR Proteins Upon EctopicExpression in APC-Like Cells, on Human T Cell Responses

The effects of LY6G6F, TMEM25 and LSR on human T cell responses wereevaluated following their ectopic expression in ‘T cell stimulator’cells: a murine thymoma cell line, Bw5147, which were engineered toexpress membrane-bound anti-human CD3 antibody fragments, that cantrigger the TCR-complex on human T cells, with or without co-expressionof putative costimulatory or coinhibitory ligands.

Codon-optimized cDNAs encoding LY6G6F (SEQ ID NO: 1), TMEM25 (SEQ ID NO:7) and LSR (SEQ ID NO: 11) were gene-synthesized and directionallycloned into a retroviral vector pCJK2 via Sfi-I sites. Monocistronicexpression constructs were generated. The constructs were validated byagarose gel electrophoresis and were expressed in Bw5147 cellsdisplaying high levels of membrane bound anti-CD3 antibody (Bw-3/2)(Leitner et al., 2010). As negative control Bw5147 cells transduced with“empty” vector (pCJK2) were used. In addition, Bw-3/2 cells expressingcostimulatory molecules (ICOSL and CD70) and Bw-3/2-cells expressingcoinhibitory molecules (B7-H3 and B7-H1/PD-L1) were also used ascontrols. Homogenously high expression of the stimulating membrane-boundanti-CD3 antibody was confirmed by FACS using a DyLight-649 anti-mouseIgG (H+L) antibody that reacts with the murine single chain antibodyexpressed on the stimulator cells. Presence and high level transcriptionof expression monocistronic constructs in the respective stimulatorcells was confirmed by qPCR.

T cells were purified from buffy coats or heparinised blood derived fromhealthy volunteer donors and the mononuclear fraction was obtained bystandard density centrifugation using Ficoll-Paque (GE-Healthcare).Untouched bulk human T cells were obtained through MACS-depletion ofCD11b, CD14, CD16, CD19, CD33 and MHC-class II-bearing cells with therespective biotinylated mAb in conjunction with paramagneticstreptavidin beads (Leitner et al., 2009). Purified CD8 T cells and CD4T cells were obtained by adding biotinylated CD4 and CD8 mAb to thepools. Naïve CD4 T cells were isolated using the Naïve CD4+T cellIsolation Kit II (Miltenyi Biotec). Following isolation, cells wereanalyzed for purity by FACS, and samples with sufficient purity (>90%)were used for the experiments.

The stimulator cells were harvested, counted, irradiated (2×3000 rad)and seeded in flat-bottom 96-well plates (20000 cells/well). Liquidnitrogen stored MACS-purified T cells were thawed, counted and added tothe wells at 100.000 cells per well; total volume was 200 μl/well.Triplicate wells were set up for each condition. Following 48 hours ofco-culture, ³H-thymidine (final concentration of 0.025 mCi;PerkinElmer/NewEngland Nuclear Corporation, Wellesley, Mass.) was addedto the wells. Following further culturing for 18 hours, the plates wereharvested on filter-plates and incorporation of ³H-Thymidine wasdetermined as described in Pfistershammer et al., 2004. In addition, aseries of experiments with MACS-purified T cell subsets (CD8 T cells,CD4 T cells, and naïve CD45RA-positive CD4 T cells) were performed.Additional controls in all experiments included wells with stimulatorcells alone to assess the cells microscopically and also to determinebasal ³H-Thymidine incorporation of the stimulator cell w/o T cells.Results with stimulator cells that quickly disintegrated followingirradiation were excluded from the analysis.

Results shown in FIG. 33 are an average of several experiments, and showthe effect of stimulator cells expressing LY6G6F, TMEM25 or LSR on theproliferation of human bulk T cells (FIG. 33A), CD4+ T cells (FIG. 33B),CD8+ T cells (FIG. 33C), or naïve CD4 CD45RA+ T cells (FIG. 33D).Expression of control costimulatory molecules (ICOSL and CD70) resultedin a consistent and pronounced stimulation of proliferation of all cellsubtypes. Similarly to expression of control coinhibitory molecules(B7-H3 and B7-H1/PD-L1), which resulted in a mild inhibition ofproliferation of different T cell subtypes, expression of LY6G6F, TMEM25and LSR also resulted in a mild inhibition of T cell proliferation, withthe most pronounced inhibitory effects exhibited on CD8+ T cells.

Example 34

Characterizing the Target Cells for LY6G6F, VSIG10, TMEM25 and/or LSRProteins by Determining their Binding Profile to Immune Cells

Splenocytes from D011.10 mice (transgenic mice in which all of the CD4+T cells express a T cell receptor that is specific for OVA323-339peptide) are activated in the presence of OVA323-339 peptide, and cellsare collected at t=0, 6, 12, 24, and 48 hours following initialactivation to determine which cell type is expressing a receptor forLY6G6F, VSIG10, TMEM25 and/or LSR over time. Cells are then co-stainedfor CD3, CD4, CD8, B220, CD19, CD11b, and CD11c.

Example 35 Assessment of the Effect of LY6G6F, VSIG10, TMEM25 or LSR ECDIg Fusion Proteins on the Ability of B Cells to Class-Switch and SecreteAntibody

Resting B cells are isolated from unprimed C57BL/6 mice and activated invitro in the presence of anti-CD40 plus (i) no exogenous cytokine, (ii)IL-4, or (iii) IFN-γ. The cell cultures receive control Ig (mIgG2a),anti-CD86 mAb (as a positive control for increased Ig production), orany one of LY6G6F, VSIG10, TMEM25 and LSR ECD fusion proteins describedin Example 5 herein, at the time of culture set up, and are cultured for5 days. The LY6G6F, VSIG10, TMEM25 and LSR ECD fusion proteins aretested at three concentrations each. At the end of culture, supernatantsare tested for the presence of IgM, IgG1, and IgG2a via ELISA. If thereappears to be an alteration in the ability of the B cells toclass-switch to one isotype of antibody versus another, then the numberof B cells that have class switched is determined via ELISPOT. If thereis an alteration in the number of antibody producing cells, then it isdetermined if there is an alteration in the level of γ1- and γ2a-steriletranscripts versus the mature transcripts for IgG1 and IgG2a.

Assessment of the Therapeutic Effect of LY6G6F, VSIG10, TMEM25 or LSRECD Ig Fusion Proteins for Treatment of Autoimmune Diseases Example 36Efficacy of LY6G6F, VSIG10, TMEM25 or LSR ECD Ig Fusion Proteins inMouse R-EAE Model of Multiple Sclerosis

The therapeutic effect of TMEM25-ECD-Ig, LSR-ECD-Ig and VSIG10-ECD-Igfusion proteins (SEQ ID NOs: 25, 26 and 24, respectively) for treatmentof autoimmune diseases was tested in a mouse model of MultipleSclerosis; Relapsing Remitting Experimental Autoimmune Encephalomyelitis(R-EAE):

Female SJL mice 6 weeks old were purchased from Harlan and maintained inthe CCM facility for 1 week prior to beginning the experiment. Mice wererandomly assigned into groups of 10 animals and primed with 50 μgPLP139-151/CFA on day 0. Mice received 6 i.p. injections of 100 ug/doseof TMEM25-ECD-Ig (SEQ ID NO: 25), LSR-ECD-Ig (SEQ ID NO: 26), mIgG2aisotype control, or CTLA4-Ig (mouse ECD fused to mouse IgG2a Fc) aspositive control. Treatments began at the time of onset of diseaseremission and were given 3 times per week for 2 weeks. Mice werefollowed for disease symptoms. On day 35, (during the peak of diseaserelapse) 5 mice of each group were assayed for DTH (delayed typehypersensitivity) response to disease inducing epitope (PLP139-151) andto relapse-associated myelin epitope (PLP178-191) via injection of 10 μgof PLP139-151 in one ear and PLP178-191 into the opposite ear. The levelof ear swelling was assayed at 24 hours post challenge.

The present Example shows a pronounced decrease in disease severity ofR-EAE-induced mice upon treatment with TMEM25-ECD-Ig (SEQ ID NO: 25) orLSR-ECD-Ig (SEQ ID NO: 26), in a therapeutic mode with 100 ug/dose at 3times per week, as shown in FIG. 34A. The level of inhibition wassimilar to that of CTLA4-Ig.

In addition, treatment of R-EAE mice with TMEM25-ECD-Ig (SEQ ID NO: 25)or LSR-ECD-Ig (SEQ ID NO: 26) dramatically inhibited DTH responses tothe disease inducing epitope (PLP139-151) and to relapse-associatedepitope (PLP178-191) at day 35 (FIG. 34B).

To test the dose dependency of the efficacy of TMEM25-ECD-Ig (SEQ ID NO:25) as well as its mode of action in the PLP-induced R-EAE model,disease was induced as described above and mice were treated from onsetof disease remission with 100, 30 or 10 ug/dose TMEM25-ECD-Ig, 3 timesper week over two weeks. TMEM25-ECD-Ig decreased the level of diseaseseverity in a dose dependent manner as shown by the milder effectobserved by the lowest dose tested (10 ug/dose), which is significantlydifferent from the effect of the high dose (100 ug/dose) (FIG. 35A).TMEM25-ECD-Ig also inhibited DTH responses to spread epitopes PLP178-191and MBP84-104 on days 45 and 76 (FIG. 35B). Furthermore, TMEM125-ECD-Iginhibited recall responses of day 45 and day 76 splenocytes and day 45cervical lymph node cells, to PLP139-151, PLP178-191 and MBP84-104(FIGS. 35C and 35D). This was manifested mainly in inhibition ofproliferation as well as reduction in IFNγ and IL-17 release.TMEM25-ECD-Ig also inhibits IL-4 and IL-10 release from cervical lymphnode cells of mice treated at 30 ug/dose TMEM25-ECD-Ig. There was noconsistent effect on IL-4 and IL-10 release from splenocytes under theseconditions.

The beneficial effect of TMEM25-ECD-Ig (SEQ ID NO: 25) in the R-EAEmodel was also accompanied by a significant reduction in theinfiltration of immune cells to the CNS (FIG. 35E). Although none of thelineages tested in the CNS was significantly changed, there was a cleartrend for reduction in CD4+ T cells and Dc (CD:11C+) and some increasein the B cell (CD19+) population, although this did not reachstatistical significance (FIG. 35E).

VSIG10-ECD-Ig (SEQ ID NO: 24) was also tested in the PLP-induced R-EAEmodel described above. Treatments began on the day of onset of remissionand given at 100 ug/dose 3×/week over 2 weeks. VSIG10-ECD-Igsignificantly reduced disease severity as manifested in reduction indisease score (FIG. 36A). The beneficial effect of VSIG10-ECD-Ig in thismodel was also accompanied by inhibition of day 45 and day 76 DTHresponses to spread epitopes PLP178-191 and to MBP84-104 (FIG. 36B). Inaddition, VSIG10-ECD-Ig (SEQ ID NO: 24) inhibited recall responses ofsplenocytes and draining (cervical) lymph node cells taken on day 45, inresponse to activation with inducing epitope PLP139-151, or spreadepitopes PLP178-191 and MBP84-104 (FIGS. 36C and 36D). This wasmanifested in inhibition of cell proliferation as well as secretion ofIFNg, IL-17, IL-4 and IL-10.

Interestingly, on day 76 VSIG10-ECD-Ig (SEQ ID NO: 24) inhibited onlyMBP84-104 induced splenocytes proliferation, but not proliferationinduced by the earlier myelin epitopes, (FIG. 36C). VSIG10-ECD-Igtreatment in the R-EAE model also significantly reduced the infiltrationof immune cells to the CNS which was accompanied by evident but notsignificant elevation in the number of cells in the lymph nodes, (FIG.36E). The major cell subtype that was reduced in the CNS was CD4+ Tcells, however, there was also a clear trend of reduction of CD19+ Bcells and CD11c+ Dcs in the CNS. All these immune cell subtypes weresignificantly elevated in the lymph nodes, suggesting that VSIG10-ECD-Igmay inhibit trafficking of immune cells from the lymph nodes to the CNS.

LY6G6F-ECD-Ig fusion protein is studied in a similar model of MultipleSclerosis.

Example 37 Efficacy of LY6G6F, VSIG10, TMEM25 or LSR ECD Ig FusionProteins in Mouse CIA Models of Rheumatoid Arthritis

Study I:

LSR-ECD-Ig (SEQ ID NO: 26) was tested in mouse model of collagen-inducedarthritis (CIA) which is a model of rheumatoid arthritis. Male DBA/1mice were housed in groups of 8-10, and maintained at 21° C.±2° C. on a12h light/dark cycle with food and water ad libitum. Arthritis wasinduced by immunisation with type II collagen emulsified in completeFreund's adjuvant. Mice were monitored on a daily basis for signs ofarthritis. On the appearance of arthritis (day 1) treatment withLSR-ECD-Ig (SEQ ID NO: 26), mIgG2a isotype control or CTLA4-Ig (mouseECD fused to mouse IgG2a Fc) as positive control (100 ug/dose, each) wasinitiated and given 3 times per week for 10 days. Hind footpad swellingwas measured (using microcalipers), as well as the number and degree ofjoint involvement in all four limbs. This yielded two measurements,clinical score and footpad thickness that can be used for statisticalassessment.

At the end of the treatment period mice were bled and sacrificed. Forhistological analysis, paws were removed at post mortem, fixed inbuffered formalin (10% v/v), then decalcified in EDTA in bufferedformalin (5.5% w/v). The tissues are then embedded in paraffin,sectioned and stained with haematoxylin and eosin. The scoring system isas follows:

0=normal; 1=synovitis but cartilage loss and bone erosions absent orlimited to discrete foci; 2=synovitis and significant erosions presentbut normal joint architecture intact; 3=synovitis, extensive erosions,joint architecture disrupted.

The present Example shows that treatment of mice with established CIAwith LSR-ECD-Ig at 100 ug/dose 3 times/week for 10 days resulted inpotent reduction of clinical score (FIG. 37A) and paw swelling (FIG.37B) and histological damage (FIG. 37C). The efficacy of LSR-ECD-Ig (SEQID NO: 26) was similar to that obtained with CTLA4-Ig.

The efficacy of TMEM25-ECD-Ig, VSIG10-ECD-Ig and LY6G6F-ECD-Ig isevaluated in this CIA model.

Treatment with TMEM25-ECD-Ig (SEQ ID NO: 25) or with LSR-ECD-Ig (SEQ IDNO: 26) did not show efficacy in a more severe CIA model in which aboost with type II collagen emulsified in complete Freund's adjuvant isgiven on day 21. In this severe CIA Enbrel, a positive control, given atthe same regimen and dosage, had very weak efficacy. Treatment withTMEM25-ECD-Ig also did not show a therapeutic effect in a CIA model witha collagen type II boost without the adjuvant given on day 21.

Study II:

The efficacy of LY6G6F ECD Ig fusion protein in the CIA model wasstudied using a modified CIA model as follows: female DBA/1 mice(Taconic Farms, 9-11 weeks old) were acclimated for 7 days. On day 0,mice were immunized with chicken collagen/CFA, 0.05 mL EK-0210emulsion/mouse (Hooke Laboratories, Inc.) and on day 20 a booster withchicken collagen/IFA, 0.05 mL EK-0211 emulsion/mouse (HookeLaboratories, Inc.) was injected. Mice were scored daily and enrolledinto one of the following treatment groups on the day of onset ofarthritis:

-   -   Group 1: LY6G6F-ECD-Ig (SEQ ID NO: 23), i.p., Q2D, 30 mg/kg for        2 wks, 10 mL/kg.    -   Group 2: Vehicle (PBS) Q2D, for 2 wks, 10 mL/kg (negative        control).

From the time of enrolment, mice were scored every other day forclinical signs and ankylosis according to the following scoring system:

Clinical Score:

0 Normal paw. 1 One toe inflamed and swollen. 2 More than one toe, butnot entire paw, inflamed and swollen, OR Mild swelling of entire paw. 3Entire paw inflamed and swollen. 4 Very inflamed and swollen paw orankylosed paw. If the paw is ankylosed, the mouse cannot grip the wiretop of the cag

Ankylosis Score:

Paw Score Clinical Observations 0 No ankylosis 1 Mild ankylosis 2Moderate ankylosis 3 Severe ankylosis

The present Example shows that treatment of mice with established CIAwith 30 mg/kg LY6G6F-ECD-Ig Q2D over 2 weeks from onset of arthritisresulted in alleviation of disease manifested in reduction of diseasescore (FIG. 38).

The efficacy of VSIG10-ECD-Ig (SEQ ID NO: 24) and TMEM25-ECD-Ig (SEQ IDNO: 25) is evaluated in a similar model.

Study III: Effect of LY6G6F, VSIG10, TMEM25 and Lsr ECD-Ig FusionProteins on Tolerance Induction in Transfer Model of CIA

To further understand the effect of LY6G6F, VSIG10, TMEM25 and LSRECD-Ig fusion proteins on immune regulation, the ability of theseproteins to induce tolerance in a transfer model of arthritis isanalysed.

In brief, spleen and LN cells from arthritic DBA/1 mice treated for 10days with LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SEQ IDNOs: 23, 24, 25 and 26, respectively) or control Ig2a are removed andinjected i.p into T-cell deficient C.B-17 SCID recipients. The mice thenreceive an injection of 100 μg type II collagen (without CFA), necessaryfor successful transfer of arthritis. Arthritis is then monitored in theSCID mice. Histology is performed and anti-collagen antibody levels aremeasured to determine that the LY6G6F, VSIG10, TMEM25 and LSR ECD Igfusion proteins treatment confers long-term disease protection.

Example 38 Assessment of the Effect of LY6G6F, VSIG10, TMEM25 and LSRECD-Ig Fusion Proteins in a Viral Infection Model of TMEV

Theiler's murine encephalomyelitis virus (TMEV) is a natural endemicpathogen of mice that causes an induced demyelinating disease (TMEV-IDD)in susceptible strains of mice (SJL/J, H-2KS) that resembles the primaryprogressive form of MS (Munz et al., Nat Rev Immunol 2009; 9:246-58).TMEV infection results in a life-long persistent virus infection of theCNS leading to development of a chronic T cell-mediated autoimmunedemyelinating disease triggered via de novo activation of CD4 T cellresponses to endogenous myelin epitopes in the inflamed CNS (i.e.epitope spreading) (Miller et al., Nat Med 1997; 3:1133-6; Katz-Levy etal., J Clin Invest 1999; 104:599-610).

SJL mice clear the majority of the virus within 21 days post infection,however a latent viral infection is maintained and infect microglia,astrocytes, and neurons. Disease symptoms are manifested around day25-30 post infection.

The effect of treatment with LY6G6F, VSIG10, TMEM25 or LSR Ig fusionproteins (SED ID NOs: 23, 24, 25 and 26, respectively) on acute andchronic phases of viral infection is studied in the TMEV-IDD model byassessment of viral clearance and disease severity.

Method:

Female SJL/J mice (5-6 weeks) are infected with TMEV by intracranialinoculation in the right cerebral hemisphere of 3×10⁷ plaque formingunits (PFU) of the BeAn strain 8386 of TMEV in 30 ul serum-free medium.From day 2 post infection mice are treated with Control Ig, LY6G6F,VSIG10, TMEM25 or LSR ECD-Ig fusion proteins, at 100 ug/dose each; 3doses/week for 2 weeks.

Mice are followed for clinical scoring. On day 7 and day 14 postinfection (after 3 and 6 treatments respectively) brains and spinalcords are collected from 5 mice in each treatment group for plaqueassays. The tissues are weighted so that the ratio of PFU/mg of CNStissue could be calculated after the plaque assay is completed.

TMEV plaque assay:

Brains and spinal cords of mice treated with Control Ig (mouse IgG2a),or with each of LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusion proteins(SED ID NOs: 23, 24, 25 and 26, respectively) are collected at days 7and 14 post-infection from non-perfused anesthetized mice. The Brainsand spinal cords are weighed, and homogenized. CNS homogenates areserially diluted in DMEM and added to tissue culture-treated plates ofconfluent BHK-21 cells for 1h incubation at room temperature, withperiodic gentle rocking.

A media/agar solution is mixed 1:1 (volume:volume), added to cells andallowed to solidify at room temperature. The plates are then cultured at34 deg C. for 5 days. At the end of culture, 1 ml of formalin is addedand incubated at room temperature for 1 h to fix the BHK monolayer. Theformalin is poured off into a waste container, and the agar is removedfrom the plates. Plaques are visualized by staining with crystal violetfor 5 min, and plates are gently rinsed with diH2O. To determine PFU/mlhomogenate, the number of plaques on each plate is multiplied by thedilution factor of the homogenate and divided by the amount ofhomogenate added per plate. The PFU/ml is divided by the weight of thetissue to calculate PFU/mg tissue.

Example 39 Assessment of the Effect of LY6G6F, VSIG10, TMEM25 and LSRECD-Ig Fusion Proteins on Primary and Secondary Immune Response to ViralInfection in a Mouse Model of Influenza

To test the effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusionproteins (SED ID NOs: 23, 24, 25 and 26, respectively) on primary andsecondary immune responses to viral infection, BALB/c naïve mice (forprimary immune responses) and ‘HA-memory mice’, is used, as well as‘polyclonal flu-memory mice’ (to assess secondary responses mediated bymemory CD4 T cells), which are generated as detailed in Teijaro et al.,J Immunol. 2009: 182; 5430-5438, and described below.

To obtain ‘HA-memory mice’, first HA-specific memory CD4 T cells aregenerated, naive CD4 T cells are purified from spleens of HA-TCR mice[BALB/c-HA mice which express transgenic T cell receptor (TCR) specificfor influenza hemagglutinin (HA) peptide (110-119)] and primed in vitroby culture with 5.0 microg/ml HA peptide and mitomycin C-treated,T-depleted BALB/c splenocytes as APCs for 3 days at 37° C. The resultantactivated HA-specific effector cells are transferred into congenicBALB/c (Thy1.1) hosts (5×10⁶ cells/mouse) to yield “HA-memory mice” witha stable population of HA-specific memory CD4 T cells.

To obtain ‘polyclonal-memory mice’, first polyclonal influenza-specificmemory CD4 T cells are generated, by infecting BALB/c mice intranasallywith a sublethal dose of PR8 influenza, CD4 T cells are isolated 2-4months postinfection, and the frequency of influenza-specific memory CD4T cells is determined by ELISPOT. CD4 T cells from previously primedmice are transferred into BALB/c hosts to generate “polyclonalflu-memory” mice with a full complement of endogenous T cells.

Primary and secondary responses to influenza virus are tested byinfecting naïve BALB/c mice or BALB/c-HA memory mice and BALB/c‘polyclonal flu-memory mice’ with sublethal or lethal doses of PR8influenza virus by intranasal administration.

Mice are treated with LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusionproteins or with mIgG2a control before and following influenzachallenge. Weight loss and mortality will be monitored daily. Six daysafter the challenge, viral content in the bronchoalveolar lavage (BAL)is analyzed by collecting lavage liquid and testing the supernatant forviral content by determining the tissue culture infectious dose 50%(TCID50) in MDCK cells. In addition, lung tissue histopathology isperformed.

To test the effect LY6G6F, VSIG10, TMEM25 and LSR ECD-Ig fusion proteinson T cell expansion BALB/c or BALB/c-HA memory mice or BALB/c‘polyclonal flu-memory mice’ are infected as above and administered withBrdU (1 mg/dose) on days 3, 4 and 5 post infection. On day 6, spleen andlung are harvested and BrdU incorporation is estimated. Cytokineproduction by lung memory CD4 T cells during influenza challenge is alsostudied in HA-specific memory CD4 T cells stimulated in vitro with HApeptide in the presence LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusionproteins or with IgG2a for 18 hours.

Example 40 Assessment of the Effect of LY6G6F, VSIG10, TMEM25 and LSRECD-Ig Fusion Proteins on Primary and Secondary CD8 T Cell Response toViral Infection in a Mouse Model of Influenza

The effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SEDID NOs: 23, 24, 25 and 26, respectively) on primary CD8 T cell responsesto influenza virus is studied according to methods as described in theliterature (Hendriks et al., J Immunol 2005; 175; 1665-1676; Bertram etal., J Immunol. 2004; 172:981-8) using C57BL/6 mice infected withinfluenza A HKx31 by intranasal or intraperitoneal administration.LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins or mIgG2a controlare administered during priming. Animal weight loss and mortality ismonitored daily. To follow virus-specific CD8+ T cells, MHC H-2Dbtetramers loaded with the major CD8 T cell epitope, the NP₃₆₆₋₃₇₄peptide are used. Virus-specific H-2D^(b)/NP366-374+ CD8+ T cells in thelung, draining lymph nodes, and spleen are expected to reach a peakaround day 8-10 post infection and decline thereafter to only 1.5%virus-specific CD8 T cells (Hendriks et al J Immunol 2005; 175;1665-1676; Bertram et al., J Immunol. 2002; 168:3777-85; Bertram et a.,J Immunol. 2004; 172:981-8). Thus, mice are sacrificed at days 8 and 21post infection, and virus-specific CD8 T cell numbers is evaluated inthe lung, draining lymph nodes and spleen. Viral clearance is assessed.CD8 T cell responses are evaluated in spleen cell suspensions, andinclude intracellular IFN-γ staining and CTL activity, as previouslydescribed (Bertram et a., J Immunol. 2004; 172:981-8) and detailedbelow.

Cells are surface-stained with FITC-conjugated anti-mouse CD62L,PE-conjugated anti-mouse CD8 to measure CD8+ activated T cells (oranti-mouse CD4 to follow CD4+ cells). In addition to these Abs,allophycocyanin-labeled tetramers consisting of murine class I MHCmolecule H-2Db, β₂-microglobulin, and influenza NP peptide, NP₃₆₆₋₃₇₄are used to measure influenza-specific CD8 T cells. For intracellularIFN-γ staining, cell suspensions are restimulated in culture medium for6 h at 37° C. with 1 μM NP₃₆₆₋₃₇₄ peptide and GolgiStop (BD PharMingen,San Diego, Calif.). Cells are then harvested, resuspended in PBS/2%FCS/azide, and surface stained with PE-anti-CD8 and FITC-anti-CD62L asdescribed above. After surface staining, cells will be fixed inCytofix/Cytoperm solution (BD PharMingen) and then stained withallophycocyanin-conjugated antimouse IFN-γ diluted in 1× perm/washsolution (BD PharMingen). Samples are analyzed by Flow Cytometry.

For cytotoxicity assays (CTL responses) splenocytes frominfluenza-infected mice are incubated for 2 h at 37° C. to removeadherent cells. Serial 3-fold dilutions of effectors are assayed foranti-influenza NP₃₆₆₋₃₇₄-specific CTL activity against ⁵¹Cr-labeled EL4cells pulsed with 50 μM NP₃₆₆₋₃₇₄ peptide for 6 h as described byBertram et al 2002 and Bertram et al 2004.

At 3 weeks postinfection, some mice are rechallenged with theserologically distinct influenza A/PR8/34 (PR8), which shares the NPgene with influenza A HKx31, but differs in hemagglutinin andneuraminidase, so that neutralizing Abs do not limit the secondary CTLresponse. Mice are sacrificed at days 5 & 7 following virus rechallenge,and virus-specific CD8 T cell numbers is evaluated in the lung, draininglymph nodes and spleen as described by Hendriks et al and Bertram et al(Hendriks et al., J Immunol 2005; 175; 1665-1676; Bertram et al., JImmunol. 2004; 172:981-8) and detailed above. Secondary CD8 T cellresponses, including intracellular IFN-γ staining and CTL activity, areevaluated in spleen cell suspensions of mice at days 5 & 7 followingvirus rechallenge, as described above.

To determine the effect of LY6G6F, VSIG10, TMEM25 and LSR ECD-Ig fusionproteins on expansion and accumulation of memory CD8+ T cells during thesecondary response, adoptive transfer experiments are performed,according to methods previously described (Hendriks et al., J Immunol2005; 175; 1665-1676; Bertram et al., J Immunol. 2004; 172:981-8): miceare immunized with influenza influenza A HKx31. Twenty-one days later, Tcells are purified from spleens on mouse T cell enrichment immunocolumns(Cedarlane Laboratories, Hornsby, Ontario, Canada) and labeled with CFSE(alternatively Thy1.1 congenic mice are used as recipients). Equalnumbers of tetramer-positive T cells are injected through the tail veinof recipient mice. Mice are rechallenged with influenza virus asdescribed above, and 7 days later splenocytes are evaluated for donorvirus-specific CD8 T cells, as detailed above.

Example 41 Assessment of Protein Expression in Exhausted T Cells, andthe Binding and Effect of the LY6G6F, VSIG10, TMEM25 and LSR ECD-IgFusion Proteins on Reversing Exhausted T Cell Phenotype

Memory CD8 T-cell differentiation proceeds along distinct pathways afteran acute versus a chronic viral infection (Klenerman and HillNat Immunol6, 873-879, 2005). Memory CD8 T cells generated after an acute viralinfection are highly functional and constitute an important component ofprotective immunity. In contrast, chronic infections are oftencharacterized by varying degrees of functional impairment ofvirus-specific T-cell responses, and this defect is a principal reasonfor the inability of the host to eliminate the persisting pathogen.Although functional effector T cells are initially generated during theearly stages of infection, they gradually lose function during thecourse of the chronic infection leading to exhausted phenotypecharacterized by impaired T cell functionality.

Study I. The Effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig FusionProteins on Clearance of Viral Infection and on T Cell Functions DuringAcute and Chronic Viral Infection.

The effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SEDID NOs: 23, 24, 25 and 26, respectively) on acute and chronic viralinfection is evaluated in a mouse model of infection with LCMV(lymphocytic chroriomeningitis virus) according to methodology describedby Wherry et al J. Virol. 77: 4911-4927, 2003 and Barber et al Nature,2006, and detailed below.

Two LCMV strains that can cause either acute or chronic infections inadult mice are used; the Armstrong strain which is cleared within aweek, and the clone 13 strain which establishes a persistent infectionthat can last for months. As these two strains differ in only two aminoacids, preserving all known T cell epitopes, it is possible to track thesame CD8 T cell responses after an acute or chronic viral infection. Incontrast to the highly robust memory CD8 T cells generated after anacute Armstrong infection, LCMV-specific CD8 T cells become exhaustedduring a persistent clone 13 infection (Wherry et al J. Virol. 77:4911-4927, 2003; Barber et al., Nature. 2006; 439:682-7).

Mice are infected with 2×10⁵ PFU of Armstrong strain of LCMVintraperitoneally to initiate acute infection or 2×10⁶ PFU of C1-13intravenously to initiate chronic infection. Mice are treated i.p. withLY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins or with mIgG2acontrol, and with specific anti-LY6G6F, anti-VSIG10, anti-TMEM25, antiLSR—antibody or an isotype control.

The mice are monitored for numbers of virus specific CD8 T cells in thespleen, using virus-specific MHC tetramer epitopes, such as D⁶NP₃₉₆₋₄₀₄and D⁶GP₃₃₋₄₁ which differ in acute or chronic infections. CD8 T cellfunctional assays, such as intracellular cytokines levels and CTLactivity, are carried out as described by Wherry et al J. Virol. 77:4911-4927, 2003, and similarly to those described in Example 40.Additional assays include production by splenocytes after stimulationwith virus specific epitopes; and assessment of viral titers in theserum and in the spleen, liver, lung and kidney (Wherry et al J. Virol.77: 4911-4927, 2003; Barber et al., Nature. 2006; 439:682-7).

Study II.

Assessment of LY6G6F, VSIG10, TMEM25 and LSR expression on exhausted Tcells and binding of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusionproteins to exhausted T cells in order to evaluate regulation of theseproteins or their counterpart receptors during exhaustion of T cells:

T cells are isolated from mice with chronic LCMV infection induced withC1-13 strain. The cells are co-stained with fluorescently labeledanti-PD-1 Ab as positive control (PD-1 is highly expressed by exhaustedT cells) and biotinylated LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusionproteins or biotinylated anti-LY6G6F, anti-VSIG10, anti-TMEM25 andanti-LSR fusion proteins antibodies, and respective isotype control.Binding is detected by FACS analysis using fluorescently labeledstreptavidin.

Example 42

Assessment of LY6G6F, VSIG10, TMEM25 and/or LSR Protein Expression inFollicular Helper T (Tfh) Cells and the Binding of Ig Fusion Proteins toTfh Cells

Follicular helper T (Tfh) cells are a subset of CD4+ T cells specializedin B cell help (reviewed by Crotty, Annu. Rev. Immunol. 29: 621-663,2011). Tfh cells migrate into B cell follicles within lymph nodes, andinteract with cognate B cells at the T cell-B cell border andsubsequently induce germinal center B cell differentiation and germinalcenter formation within the follicle (Reviewed by Crotty, Annu. Rev.Immunol. 29: 621-663, 2011). The requirement of Tfh cells for B cellhelp and T cell-dependent antibody responses, indicates that this celltype is of great importance for protective immunity against varioustypes of infectious agents, as well as for rational vaccine design.

Tfh cells are readily identifiable at the peak of the CD4+ T cellresponse to an acute lymphocytic choriomeningitis virus (LCMV) infectionas CXCR5^(hi)SLAM¹⁰ BTLA^(hi)PD1^(hi)Bc16⁺ virus-specific CD4+ T cells(Choi et al 2011, Immunity 34: 932-946). T cells are isolated from micewith acute LCMV infection induced with 2×10⁵ PFU of Armstrong strain ofLCMV administered intraperitoneally. The cells are co-stained withfluorescently labeled antibodies for markers of Tfh (CXCR5, PD1, BTLA,Bc16) which are highly expressed by Tfh cells, and biotinylated LY6G6F,VSIG10, TMEM25 and LSR ECD-Ig fusion proteins or biotinylated antibodiesspecific for LY6G6F, VSIG10, TMEM25 and LSR, and respective isotypecontrols. Binding of Fc fused protein or antibody is detected by FACSanalysis using fluorescently labeled streptavidin.

Example 43 Assessment of the Effect of LY6G6F, VSIG10, TMEM25 and LSR IgFusion Proteins on Follicular Helper T (Tfh) Cells Generation andActivity

In order to investigate the effect of LY6G6F, VSIG10, TMEM25 and LSR ECDIg fusion proteins on Tfh differentiation and development of B cellimmunity in vivo, C57BL/6 are treated with LY6G6F, VSIG10, TMEM25 andLSR ECD Ig fusion proteins and an isotype control throughout the courseof an acute viral infection with Armstrong strain of LCMV (lymphocyticchoriomeningitis virus). Tfh differentiation and Bc16 protein expressionis assessed by FACS analysis as described by Eto et al 2011 (PLoS One 6:e17739). Splenocytes are analyzed 8 days following LCMV infection, Tfhgeneration)(CD44^(hi)CXCR5^(hi)SLAM¹⁰ and Bc16 expression is evaluatedby FACS analysis. In addition, the effect of LY6G6F, VSIG10, TMEM25 andLSR ECD Ig fusion proteins (SED ID NOs: 23, 24, 25 and 26, respectively)on antigen-specific B cell responses is evaluated as described by Eto etal 2011 (PLoS One 6: e17739), including titers of anti-LCMV IgG in theserum at 8 days following LCMV infection, and quantitation by FACSanalysis of plasma cell (CD138⁺IgD⁻) development at 8 dayspost-infection, gated on CD19+ splenocytes.

Example 44

The Effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig Fusion Proteins inModulation of Type 1 Diabetes in Nod Mice, CD28-KO NOD, and B7-2-KO NOD

The effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins arestudied in a widely used mouse model of type 1 diabetes: nonobesediabetic (NOD) mice which develop spontaneous In NOD mice, spontaneousinsulitis, the hallmark pathologic lesion, evolves through severalcharacteristic stages that begin with peri-insulitis and end with withinvading and destructive insulitis and overt diabetes. Peri-insulitis isfirst observed at 3-4 wk of age, invading insulitis at 8-10 wk, anddestructive insulitis appears just before the onset of clinicaldiabetes, with the earliest cases at 10-12 wk. At 20 wk of age, 70-80%of female NOD mice become diabetic (Ansari et al 2003 J. Exp. Med. 198:63-69).

Two KO mice: CD-28-KO NOD mice and B7-1/B7-2 double KO NOD mice, —whichdevelop accelerated diabetes (Lenschow et al 1996 Immunity 5: 285-293;Salomon et al 2000 Immunity 12: 431-440), are also used.

Study I:

NOD mice are treated with LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusionproteins (SED ID NOs: 23, 24, 25 and 26, respectively) early and latephases during the evolution of diabetes, before or after disease onset,to examine the effects of these compounds on disease pathogenesis and todemonstrate that such treatment reduces disease onset and amelioratespathogenesis. To study the effect on insulitis, blood glucose levels aremeasured 3 times/week, for up to 25 weeks (Ansari et al 2003 J. Exp.Med. 198: 63-69).

Mechanism of disease modification and mode of action is studied byexperimental evaluation of individual immune cell types: pancreas,pancreatic LNs and spleen will be harvested to obtain Tregs, Th subtypesand CD8 T cells, DCs and B cells. Effect on cytokines secretion fromcells isolated from pancreas, pancreatic LN and spleen is analysed,focused on IFNg, IL-17, IL-4, IL-10 and TGFb. Upon effect of the testedcompounds, the mechanism of disease modification is studied byexamination of individual immune cell types (including Tregs, Thsubtypes and CD8 T cells, DCs and B cells); cytokines (IFNg, IL-17,IL-4, IL-10 and TGFb) and histology. Histologycal analysis of thepancreas is carried out to compare the onset of insulitis, and thelymphocyte infiltration.

Study II—the Effect of LY6G6F Ig Fusion Proteins in Modulation of Type 1Diabetes in Adoptive Transfer Model

To further investigate the mode of action of the Ig fusion proteins, anadoptive transfer model of diabetes is used. T cells from diabetic orprediabetic NOD donors are transferred to NOD SCID recipient mice. Thesemice are monitored for development of diabetes. The urine glucose andblood glucose, and assess histology of the pancreas, and T cellresponses are monitored as described in the previous example.

Study III:

Diabetes is also induced by the transfer of activated CD4+CD62L+CD25−BDC2.5 T cells (transgenic for TCR recognizing islet specific peptide1040-p31 activated by incubation with 1040-p31) to NOD recipients. Miceare treated with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins,control mIgG2a or positive control. Treatments begin 1 day followingtransfer. Mice are followed for glucose levels 10-28 days post transfer(Bour-Jordan et al., J Clin Invest. 2004; 114(7):979-87).

Seven days post treatment pancreas, spleen, pancreatic LN and peripherallymph node cells are extracted and examined for different immune cellpopulations. In addition, recall responses are measured by testingex-vivo proliferation and cytokine secretion in response to p31 peptide.

LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins prevent or reducedisease onset or the severity thereof in the above studies.

Example 45 The Effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig FusionProteins in Lupus Mouse Models

Study I:

The lupus-prone mouse model, (NZB×NZW)F1 (B/W) is used. Cyclophosphamide(CTX) is the primary drug used for diffuse proliferativeglomerulonephritis in patients with renal lupus, Daikh and Wofsyreported that combination treatment with CTX and CTLA4-Ig was moreeffective than either agent alone in reducing renal disease andprolonging survival of NZB/NZW Fl lupus mice with advanced nephritis(Daikh and Wofsy, J Immunol, 166(5):2913-6 (2001)). In theproof-of-concept study, treatments with LY6G6F, VSIG10, TMEM25 or LSRECD Ig fusion proteins and CTX either alone or in combination aretested.

Blood samples are collected 3 days before the protein treatment and thenevery other week during and after treatments for plasma anti-dsDNAautoantibody analysis by ELISA. Glomerulonephritis is evaluated byhistological analysis of kidneys. Proteinuria is measured by testingfresh urine samples using urinalysis dipsticks.

LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SED ID NOs: 23,24, 25 and 26, respectively) have a beneficial effect in at leastameliorating lupus nephtiris.

Study II:

The NZM2410-derived B6.Sle1.S1e2.S1e3 mouse model of SLE is used.NZM2410 is a recombinant inbred strain produced from NZB and NZW thatdevelops a highly penetrant lupus-like disease with an earlier onset ofdisease (Blenman et al 2006 Lab. Invest. 86: 1136-1148). The effect ofLY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins is studied in thismodel by assessment of proteinuria and autoantibodies as describedabove.

Study III: An induced lupus model is used. This model is based onchronic graft-vs-host (cGVH) disease induced by the transfer ofIa-incompatible spleen cells from one normal mouse strain (such asB6.C-H2(bm12)/KhEg (bm12)) to another (such as C57BL/6), which causes anautoimmune syndrome resembling systemic lupus erythematosus (SLE),including anti-double-stranded DNA (anti-dsDNA) autoantibodies andimmune complex-type proliferative glomerulonephritis (Appleby et alClin. Exp. Immunol. 1989 78: 449-453); Eisenberg and Choudhury 2004Methods Mol. Med. 102:273-284). Lupus is induced in this model followinginjection of spleen cells from bm12 mice into C57BL/6 recipients. Theeffect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins isstudied in this model by assessment of proteinuria and autoantibodies asdescribed above. T cell and responses B cell responses will also beevaluated.

Study IV:

The MRL/lpr lupus prone mouse model is used. The effect of LY6G6F,VSIG10, TMEM25 and LSR ECD Ig fusion proteins is studied in this modelby assessment of proteinuria and autoantibodies as described above.

Example 46 The Effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig FusionProteins in the Control of Intestinal Inflammation.

An adoptive transfer mouse model of colitis in mice is used, wherebyTransfer of CD45RB^(high)-CD4+ naïve T cells from BALB/c mice tosyngeneic SCID mice leads to the development of an IBD-like syndrome by6-10 wks after T cell reconstitution, similar to human Crohn's disease.

SCID mice are reconstituted by i.p. injection of syngeneicCD45RB^(high)-CD4⁺ T cells either alone or cotransferred with syngeneicCD45RB^(low)-CD4⁺ or CD25⁺CD4⁺ cells (4×10⁵/mouse of each cellpopulation) (Liu et al., J Immunol. 2001; 167(3): 1830-8). Colitic SCIDmice, reconstituted with syngeneic CD45RB^(high) CD4⁺ T cells fromspleen of normal mice, are treated i.p. with LY6G6F, VSIG10, TMEM25 orLSR ECD Ig fusion proteins or Ig isotype control, twice a week startingat the beginning of T cell transfer up to 8 wk. All mice are monitoredweekly for weight, soft stool or diarrhea, and rectal prolapse. All miceare sacrificed 8 wk after T cell transfer or when they exhibit a lossof. 20% of original body weight. Colonic tissues are collected forhistologic and cytologic examinations. LY6G6F, VSIG10, TMEM25 and LSRECD Ig fusion proteins have a beneficial effect in at least amelioratinginflammatory bowel disease.

Example 47 The Effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig FusionProteins in Mouse Model of Psoriasis Study I: Establishment of PsoriasisSCID Xenograft Model.

Human psoriasis plaques are transplanted on to the SCID mice. Shavebiopsies (2.5_2.5 cm) are taken from patients with generalized plaquepsoriasis involving 5-10% of the total skin that did not receive anysystemic treatment for psoriasis or phototherapy for 6 months and didnot receive any topical preparations other than emollients for 6 weeks.The biopsies are obtained from active plaques located on the thigh orarm. Each piece of biopsy is divided into four equal parts ofapproximately 1 cm2 size. Each piece is transplanted to a separatemouse.

Under general anesthesia, a graft bed of approximately 1 cm2 is createdon the shaved area of the back of a 7- to 8-week-old CB17 SCID mouse byremoving a full-thickness skin sample, keeping the vessel plexus intacton the fascia covering the underlying back muscles. The partialthickness human skin obtained by shave biopsy is then orthotopicallytransferred onto the graft bed. Nexaband, a liquid veterinary bandage(Veterinary Products Laboratories, Phoenix, Ariz.) is used to attach thehuman skin to the mouse skin and an antibiotic ointment (bacitracin) isapplied. Mice are treated intraperitoneally three times per week for 4weeks with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins, isotypecontrol or CTLA4-Ig (positive control).

Punch biopsies (2 mm) are obtained on day 0 (before treatment) and day28 (after treatment) of the study period. Biopsies are snap frozen andcryosections for histopathological and immunohistochemical studies.Therapeutic efficacy is determined by comparing pre- and post treatmentdata: (i) rete peg lengths to determine the effect on epidermalthickness and (ii) the level of lymphomononuclear cell infiltrates todetermine the effect on inflammatory cellular infiltrates. (Raychaudhuriet al. 2008, J Invest Dermatol.;128(8):1969-76; Boehncke et al., 1999Arch Dermatol Res 291:104-6).

LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SED ID NOs: 23,24, 25 and 26, respectively) have a beneficial effect in at leastameliorating psoriasis.

Study II: The Effect of LY6G6F, VSIG10, TMEM25 and LSR in Psoriasis andColitis Model by Adoptive Transfer of CD45RB_(HI) CD4+ T Cells in ScidMice

Immunocompromised mice are injected intraveneously (i.v.) with 0.3_10⁶CD4+CD45RBhi cells. On the day following the adoptive transfer of cells,mice are injected intraperitoneally (i.p.) with 10 microg ofstaphylococcal enterotoxin B (Davenport et al., Int Immunopharmacol.2002 April; 2(5):653-72). Recipient mice are treated with LY6G6F,VSIG10, TMEM25 or LSR ECD-Ig fusion proteins (SED ID NOs: 23, 24, 25 and26, respectively), isotype control or CTLA4-Ig (positive control). Miceare evaluated once a week for 8 weeks for weight loss and presence ofskin lesions.

Obtained results are similar to those described above.

Example 48 The Effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig FusionProteins in Modulating Transplant Rejection. Study I: The Effect ofLY6G6F, VSIG10, TMEM25 and LSR in a Model of Allogeneic IsletTransplantation in Diabetic Mice.

To test the effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusionproteins (SED ID NOs: 23, 24, 25 and 26, respectively) on transplantrejection, a model of allogeneic islet transplantation is used. Diabetesis induced in C57BL/6 mice by treatment with streptozotocin. Seven dayslater, the mice are transplanted under the kidney capsule withpancreatic islets which are isolated from BALB/c donor mice. Recipientmice are treated with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusionproteins or with mIgG2a as a negative control. Tolerance with ECDI-fixeddonor splenocytes is used as the positive control for successfulmodulation islet graft rejection. Recipient mice are monitored for bloodglucose levels as a measure of graft acceptance/rejection (Luo et al.,PNAS, Sep. 23, 2008_(—) vol. 105_(—) no. 38_(—) 14527-14532).

Study II: The Effect of LY6G6F, VSIG10, TMEM25 and LSR in the HYA-Modelof Skin Graft Rejection.

In humans and certain strains of laboratory mice, male tissue isrecognized as non-self and destroyed by the female immune system viarecognition of histocompatibility-Y chromosome encoded antigens (Hya).Male tissue destruction is thought to be accomplished by cytotoxic Tlymphocytes in a helper-dependent manner.

To test the effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusedproteins (SED ID NOs: 23, 24, 25 and 26, respectively) ontransplanatation, the Hya model system is used, in which female C57BL/6mice receive tail skin grafts from male C57BL/6 donors.

In this study, female C57BL/6 mice are engrafted with orthotopicsplit-thickness tail skin from age matched male C57BL/6 mice. The miceare treated with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins,isotype control mIgG2a. Immunodominant Hya-encoded CD4 epitope (Dby)attached to female splenic leukocytes (Dby-SP) serve as positive controlfor successful modulation of graft rejection (Martin et al., J Immunol.2010 Sep. 15; 185(6): 3326-3336). Skin grafts are scored daily foredema, pigment loss and hair loss. Rejection is defined as complete hairloss and more than 80% pigment loss.

In addition, T cell recall responses of cells isolated from spleens anddraining lymph nodes at different time points are studied in response toCD4 specific epitope (Dby), CD8 epitopes (Uty and Smcy) or irrelevantpeptide (OVA 323-339) while anti CD3 stimulation is used as positivecontrol for prolifereation and cytokine secretion.

Study III:

The effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins ongraft rejection is studied in a murine model of syngeneic bone marrowcells transplantation using the Hya model system described above. Malehematopoietic cells expressing the CD45.1 marker are transplanted tofemale host mice which express the CD45.2 congenic marker. Female hostsare treated with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins orwith isotype control mIgG2a. The female hosts are followed over time andthe presence of CD45.1+ cells is monitored.

Example 49

Establishment of the Role of LY6G6F, VSIG10, TMEM25 and/or LSR ProteinsAccording to at Least Some Embodiments of the Invention as Modulators ofCancer Immune Surveillance:

1) In Vivo Proof of Concept

a) Mouse Cancer Syngeneic Model:

(i) Tumor cells, over expressing any one of LY6G6F, VSIG10, TMEM25and/or LSR proteins or a non-relevant control protein are transplantedto genetically matched mice. Tumor volume (and tumor weight aftersacrificing the animals) and ex vivo analysis of immune cells from tumordraining lymph nodes or spleens are then examined to demonstrate therejection of the tumor to be delayed (i.e. tumor over expressing LY6G6F,VSIG10, TMEM25 and/or LSR grow faster than tumors over expressing thenon-relevant control protein). Ex vivo analysis of immune cells fromtumor draining lymph nodes is expected to reveal an increase in thefrequency of regulatory T cells and a decrease in the responsiveness ofeffector T cells to stimulation. (J. Exp. Med. 2011 Vol. 208 No. 3577-592).

(ii) In vivo syngeneic model using the extra cellular domain of themouse orthologs of any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteinfused to an antibody Fc fragment (mouse ECD-Fc) (SEQ ID NO: 23, 24, 25and 26, respectively) is tested as follows. The mouse ECD-FC is injectedIV to C57BL/6 mice at 3-4 day intervals, after tumor establishment, asdescribed in J immunol 2010; 185; 2747-2753. Tumor volume (and tumorweight after sacrificing the animals) and ex vivo analysis of immunecells from tumor draining lymph nodes or spleens are then examined. As aresult of IV treatment with Mouse ECD-FC of LY6G6F, VSIG10, TMEM25and/or LSR the rejection of the tumor is delayed (i.e. in mice treatedwith the Mouse ECD-FC of LY6G6F, VSIG10, TMEM25 and/or LSR tumors growfaster than tumors in mice treated with non-relevant control protein).Ex vivo analysis of immune cells from tumor draining lymph nodes revealan increase in the frequency of regulatory T cells and a decrease in theresponsiveness of effector T cells to stimulation.

(iii) Establishment of a syngeneic tumor and treat with neutralizingantibodies directed against any one of LY6G6F, VSIG10, TMEM25 and/or LSRprotein (1, 3, 5, 7, 11, 143, 13, 15-17, 18, 28, 29-32). Tumor cells aretransplanted to genetically identical mice. After the establishment oftumors, mice are injected IV with different doses of neutralizingantibodies aimed against any one of LY6G6F, VSIG10, TMEM25 and/or LSRprotein. As a result of IV treatment with neutralizing antibodiesspecific for any one of LY6G6F, VSIG10, TMEM25 and/or LSR protein therejection of the tumor is increased (i.e. in mice treated withneutralizing antibodies against any one of LY6G6F, VSIG10, TMEM25 and/orLSR protein tumors grow slower than tumors in mice treated withnon-relevant antibody). Ex vivo analysis of immune cells from tumordraining lymph nodes reveal a decrease in the frequency of regulatory Tcells and an increase in the responsiveness of effector T cells tostimulation.

b) Human Cancer Xenograft Model:

(i) Reconstitution of the tumor immune response in a model of immunecompromised NOD.Cg-Prkdcscid Il2rgtm1Wj1/SzJ mice (Jackson lab), “NSG”mice. Human tumor is established in NSG model, and APCs pre-loaded withTumor antigens, or/and T cells (CD8 T cells pre-activated with cancertarget cells are transferred into tumor bearing NSG mice (all cellstransplanted/injected originate from cancer patients). This modelconsists of four arms: 1. APC's over expressing any one of LY6G6F,VSIG10, TMEM25 and/or LSR proteins, 2. silencing of any one of LY6G6F,VSIG10, TMEM25 and/or LSR proteins (either siRNA or ShRNA) on APC's, 3.Cancer cells over expressing any one of LY6G6F, VSIG10, TMEM25 and/orLSR proteins and 4. Silencing of any one of LY6G6F, VSIG10, TMEM25and/or LSR proteins (either siRNA or ShRNA) on cancer cells. Positive(e.g. B7-H1, PD-L1) and negative (e.g. Vector and cells alone) controlsare included. Tumor volume or tumor metastasis and mouse survival arethen examined (J. Exp. Med.; 2006; Vol. 203; p. 871-881). Overexpression of any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteinseither on APC's or on Tumor cells, lead to delayed rejection of thetumor (i.e. in mice treated with the APC's or tumor cells overexpressing any of LY6G6F, VSIG10, TMEM25 and/or LSR tumors grow fasterthan tumors in mice treated with non-relevant control protein).Silencing (with SiRNA or SHRNA) of any of LY6G6F, VSIG10, TMEM25 and/orLSR either on APC's or on tumor cells lead to enhanced rejection of thetumor.

(ii) Establishment of the NSG cancer Xenograft as described above(without genetic manipulation of APC's and/or cancer cells) andtreatment with neutralizing antibodies directed against the any one ofLY6G6F, VSIG10, TMEM25 and/or LSR proteins. Treatment of the NSGXenograft model with neutralizing antibodies directed against any ofLY6G6F, VSIG10, TMEM25 and/or LSR is gives rise to enhanced rejection ofthe tumor.

2) In Vitro Validation of Natural Killer (NK) Cell Activity

a) Binding Assay:

(i) Binding assay with human LY6G6F, VSIG10, TMEM25 and/or LSR ECD-FCproteins on activated primary-culture NK cells is performed as describedin J Immunol 2005; 174; 6692-6701.If the counter receptor of LY6G6F,VSIG10, TMEM25 and/or LSR is expressed on NK cells, binding of LY6G6F,VSIG10, TMEM25 and/or LSR ECD-Fc is observed.

(ii) Binding assay with a specific antibody directed against the any oneof LY6G6F, VSIG10, TMEM25 and/or LSR proteins on activatedprimary-culture NK cells is performed as described in PNAS, 2009, vol.109; 17858-17863. If any one of LY6G6F, VSIG10, TMEM25 and/or LSR isexpressed on NK cells, binding of LY6G6F, VSIG10, TMEM25 and/or LSRspecific antibody, respectively, is observed.

(iii) Binding assay with human LY6G6F, VSIG10, TMEM25 and/or LSR ECD-FCproteins on various human cancer cell lines that may serve as targetcells for NK killing is performed as described in J Immunol 2006; 176;6762-6769. If the counter receptor of any one of LY6G6F, VSIG10, TMEM25and/or LSR is expressed on the cancer target cells, binding of LY6G6F,VSIG10, TMEM25 and/or LSR ECD-Fc, respectively is observed.

b) Functional Killing Assay:

(i) Killing assays are performed using an over expression system (eitherNK cells or cancer target cells, over expressing any one of LY6G6F,VSIG10, TMEM25 and/or LSR proteins). The NK cells (effector; e) areco-incubated with radioactive (S35) labeled cancer target cells (target;t) in various e: t ratios, as described in PNAS, 2009, vol. 109;17858-17863. Lysis of target cells by NK killing activity is thenevaluated by measurement of radioactive emission. Over expression of anyone of LY6G6F, VSIG10, TMEM25 and/or LSR proteins on the target cancercells and/or the NK cell lines lead to down regulation of the NKmediated killing activity.

(ii) Killing assays are performed in the presence of the human LY6G6F,VSIG10, TMEM25 and/or LSR ECD-FC proteins, as described in PLoS ONE;2010; Vol. 5; p. 1-10. Treatment with the ECD-Fc of any of LY6G6F,VSIG10, TMEM25 and/or LSR interfere with the interaction of LY6G6F,VSIG10, TMEM25 and/or LSR with their counter receptors and thus decreasetheir inhibitory activity, giving rise to enhanced killing activity.

(iii) Killing assays are performed in the presence of a neutralizingantibody directed against any one of LY6G6F, VSIG10, TMEM25 and/or LSRproteins, as described in PNAS, 2009, vol. 109; 17858-17863. Treatmentwith neutralizing antibodies directed towards any of LY6G6F, VSIG10,TMEM25 and/or LSR, give rise to enhanced NK killing activity.

(iv) “Re-directed killing assay” is performed as follows: cancer targetcells expressing high density Fc receptors are coated with activatingantibodies directed against any one of LY6G6F, VSIG10, TMEM25 and/or LSRproteins and exposed to NK cells (expressing the designated LY6G6F,VSIG10, TMEM25 and/or LSR protein), as described in PNAS, 2009, vol.109; 17858-17863. Cross linking of any one of LY6G6F, VSIG10, TMEM25and/or LSR with activating antibodies give rise to reduced NK mediatedkilling activity.

3) Expression Analysis

a) Expression of LY6G6F, VSIG10, TMEM25 and/or LSR Proteins on CellsIsolated from Human Tumor Biopsies

i) Expression validation of any one of LY6G6F, VSIG10, TMEM25 and/or LSRproteins using specific antibodies directed against the any one ofLY6G6F, VSIG10, TMEM25 and/or LSR proteins, respectively, is carried outon separated cell populations from the tumor. Various cell populationsare freshly isolated from tumor biopsies (e.g. Tumor cells, endothelia,tumor associated macrophages (TAMs) and DCs, B cells and different Tcells (CD4, CD8 and Tregs) as described in J. Exp. Med.; 2006; Vol. 203;p. 871-881 and Cancer res. 2007; 67; 8900-8905, to demonstrateexpression of any of LY6G6F, VSIG10, TMEM25 and/or LSR in tumor cellsand on tumor stroma and immune infiltrate.

ii) Binding assay is performed with the human LY6G6F, VSIG10, TMEM25and/or LSR ECD-FC proteins on separated cell populations from the tumor.Isolate various cell populations from tumor biopsies (e.g. Tumor cells,endothelia, tumor associated macrophages (TAMs) and DCs, B cells anddifferent T cells (CD4, CD8 and Tregs) freshly isolated from tumors asdescribed in J. Exp. Med.; 2006; Vol. 203; p. 871-881 and Cancer res.2007; 67; 8900-8905, to show expression of the counter receptor for anyof LY6G6F, VSIG10, TMEM25 and/or LSR in tumor cells and on tumor stromaand immune cells.

b) Expression of LY6G6F, VSIG10, TMEM25 and/or LSR Proteins on CellsIsolated from Draining Lymph Nodes and Spleens of Tumor Bearing Mice

(i) Expression validation of LY6G6F, VSIG10, TMEM25 and/or LSR proteinsusing specific antibodies directed against LY6G6F, VSIG10, TMEM25 and/orLSR proteins, respectively, is done on epithelial cancer cells as wellas on immune cells from tumor draining lymph nodes vs. spleen of tumorbearing C57 mice, as described in Clinical Cancer Research 1996 Vol. 2,811-820. Three different cancer types are being tested: B16 (melanoma),ID8 (ovarian) and MC38 (colon)), to show expression of any of LY6G6F,VSIG10, TMEM25 and/or LSR in tumor cells and in immune cells in thetumor draining lymph node.

ii) Binding assay with mouse LY6G6F, VSIG10, TMEM25 and/or LSR ECD-FCproteins on cells isolated from epithelial cancer as well as on immunecells from tumor draining lymph nodes versus spleen of tumor bearing C57mice, is carried out as described above, to show show expression of thecounter receptor for any of LY6G6F, VSIG10, TMEM25 and/or LSR in tumorcells and in immune cells in the tumor draining lymph node.

c) Expression of LY6G6F, VSIG10, TMEM25 and/or LSR Proteins on M2Polarized Macrophages

-   -   (i) Expression validation of LY6G6F, VSIG10, TMEM25 and/or LSR        proteins using specific antibodies directed against LY6G6F,        VSIG10, TMEM25 and/or LSR proteins, respectively, is done on        primary monocytes isolated from peripheral blood, differentiated        into macrophages and exposed to “M2 driving stimuli” (e.g. IL4,        IL10, Glucocorticoids, TGF beta), as described in Nat. Immunol.        2010; Vol. 11; p. 889-896, to show expression of any of LY6G6F,        VSIG10, TMEM25 and/or LSR in M2 differentiated Macrophages.    -   ii) Binding assay with LY6G6F, VSIG10, TMEM25 and/or LSR human        ECD-FC proteins on primary monocytes isolated from peripheral        blood, differentiated into macrophages and exposed to “M2        driving stimuli” (e.g. IL4, IL10, Glucocorticoids, TGF beta) is        carried out as described above, to show expression of the        counter receptor for any of LY6G6F, VSIG10, TMEM25 and/or LSR in        M2 differentiated Macrophages.

Example 50

Development of Fully Human Anti-LY6G6F, Anti-VSIG10, Anti-TMEM25 and/orAnti-LSR Antibodies

Generation Of Human Monoclonal Antibodies Against LY6G6F, VSIG10, TMEM25and/or LSR Antigen

Fusion proteins composed of the extracellular domain of the LY6G6F,VSIG10, TMEM25 and/or LSR linked to a mouse IgG2 Fc polypeptide aregenerated by standard recombinant methods and used as antigen forimmunization.

Transgenic HuMab Mouse.

Fully human monoclonal antibodies to LY6G6F, VSIG10, TMEM25 and/or LSRare prepared using mice from the HCo7 strain of the transgenic HuMabMouse®, which expresses human antibody genes. In this mouse strain, theendogenous mouse kappa light chain gene has been homozygously disruptedas described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenousmouse heavy chain gene has been homozygously disrupted as described inExample 1 of PCT Publication WO 01/09187. Furthermore, this mouse straincarries a human kappa light chain transgene, KCo5, as described inFishwild et al. (1996) Nature Biotechnology 14:845-851, and a humanheavy chain transgene, HCo7, as described in U.S. Pat. Nos. 5,545,806;5,625,825; and 5,545,807.

HuMab Immunizations:

To generate fully human monoclonal antibodies to LY6G6F, VSIG10, TMEM25and/or LSR, mice of the HCo7 HuMab Mouse strain can be immunized withpurified recombinant LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteinderived from mammalian cells that are transfected with an expressionvector containing the gene encoding the fusion protein. Generalimmunization schemes for the HuMab Mouse are described in Lonberg, N. etal (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851 and PCT Publication WO 98/24884. The mice are6-16 weeks of age upon the first infusion of antigen. A purifiedrecombinant LY6G6F, VSIG10, TMEM25 and/or LSR antigen preparation (5-50.mu.g, purified from transfected mammalian cells expressing LY6G6F,VSIG10, TMEM25 and/or LSR fusion protein) is used to immunize the HuMabmice intraperitoneally.

Transgenic mice are immunized twice with antigen in complete Freund'sadjuvant or Ribi adjuvant IP, followed by 3-21 days IP (up to a total of11 immunizations) with the antigen in incomplete Freund's or Ribiadjuvant. The immune response is monitored by retroorbital bleeds. Theplasma is screened by ELISA (as described below), and mice withsufficient titers of anti-LY6G6F, VSIG10, TMEM25 and/or LSR humanimmunoglobulin are used for fusions. Mice are boosted intravenously withantigen 3 days before sacrifice and removal of the spleen.

Selection of HuMab mice producing anti-LY6G6F, anti-VSIG10, anti-TMEM25and/or anti-LSRAntibodies:

To select HuMab mice producing antibodies that bind LY6G6F, VSIG10,TMEM25 and/or LSR sera from immunized mice is tested by a modified ELISAas originally described by Fishwild, D. et al. (1996). Briefly,microtiter plates are coated with purified recombinant LY6G6F, VSIG10,TMEM25 and/or LSR fusion protein at 1-2 .mu.g/ml in PBS, 50 .mu.l/wellsincubated 4 degrees C. overnight then blocked with 200 .mu.l/well of 5%BSA in PBS. Dilutions of plasma from LY6G6F, VSIG10, TMEM25 and/orLSR-immunized mice are added to each well and incubated for 1-2 hours atambient temperature. The plates are washed with PBS/Tween and thenincubated with a goat-anti-human kappa light chain polyclonal antibodyconjugated with alkaline phosphatase for 1 hour at room temperature.After washing, the plates are developed with pNPP substrate and analyzedby spectrophotometer at OD 415-650. Mice that developed the highesttiters of anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSRantibodies are used for fusions. Fusions are performed as describedbelow and hybridoma supernatants are tested for anti-LY6G6F,anti-VSIG10, anti-TMEM25 and/or anti-LSR activity by ELISA.

Generation Of Hybridomas Producing Human Monoclonal Antibodies ToLY6G6F, VSIG10, TMEM25 and/or LSR.

The mouse splenocytes, isolated from the HuMab mice, are fused with PEGto a mouse myeloma cell line based upon standard protocols. Theresulting hybridomas are then screened for the production ofantigen-specific antibodies. Single cell suspensions of spleniclymphocytes from immunized mice are fused to one-fourth the number ofP3X63 Ag8.6.53 (ATCC CRL 1580) nonsecreting mouse myeloma cells with 50%PEG (Sigma). Cells are plated at approximately 1×10-5/well in flatbottom microtiter plate, followed by about two week incubation inselective medium containing 10% fetal calf serum, supplemented withorigen (IGEN) in RPMI, L-glutamine, sodium pyruvate, HEPES, penicillin,streptamycin, gentamycin, 1×HAT, and beta-mercaptoethanol. After 1-2weeks, cells are cultured in medium in which the HAT is replaced withHT. Individual wells are then screened by ELISA (described above) forhuman anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSRmonoclonalIgG antibodies. Once extensive hybridoma growth occurred, medium ismonitored usually after 10-14 days. The antibody secreting hybridomasare replated, screened again and, if still positive for human IgG,anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSR monoclonalantibodies are subcloned at least twice by limiting dilution. The stablesubclones are then cultured in vitro to generate small amounts ofantibody in tissue culture medium for further characterization.

Hybridoma clones are selected for further analysis.

Structural Characterization of Desired Anti-LY6G6F, Anti-VSIG10,Anti-TMEM25 and/or Anti-LSR Human Monoclonal Antibodies

The cDNA sequences encoding the heavy and light chain variable regionsof the obtained anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/oranti-LSRmonoclonal antibodies are obtained from the resultanthybridomas, respectively, using standard PCR techniques and aresequenced using standard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variableregion and of the light chain variable region are identified. Thesesequences may be compared to known human germline immunoglobulin lightand heavy chain sequences and the CDRs of each heavy and light of theobtained anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSR sequencesidentified.

Characterization of Binding Specificity and Binding Kinetics ofAnti-LY6G6F, Anti-VSIG10, Anti-TMEM25 and/or Anti-LSR Human MonoclonalAntibodies

The binding affinity, binding kinetics, binding specificity, andcross-competition of anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/oranti-LSRantibodies are examined by

Biacore analysis. Also, binding specificity is examined by flowcytometry.

Binding Affinity and Kinetics

Anti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSR antibodiesproduced according to the invention are characterized for affinities andbinding kinetics by Biacore analysis (Biacore AB, Uppsala, Sweden).Purified recombinant human LY6G6F, VSIG10, TMEM25 and/or LSR fusionprotein is covalently linked to a CMS chip (carboxy methyl dextrancoated chip) via primary amines, using standard amine coupling chemistryand kit provided by Biacore. Binding is measured by flowing theantibodies in HBS EP buffer (provided by BIAcore AB) at a concentrationof 267 nM at a flow rate of 50 .mu.l/min. The antigen-associationantibodies association kinetics is followed for 3 minutes and thedissociation kinetics is followed for 7 minutes. The association anddissociation curves are fit to a 1:1 Langmuir binding model usingBIAevaluation software (Biacore AB). To minimize the effects of avidityin the estimation of the binding constants, only the initial segment ofdata corresponding to association and dissociation phases are used forfitting.

Epitope Mapping of Obtained Anti-LY6G6F, Anti-VSIG10, Anti-TMEM25 and/orAnti-LSR Antibodies

Biacore is used to determine epitope grouping of anti-LY6G6F,anti-VSIG10, anti-TMEM25 and/or anti-LSR HuMAbs. Obtained anti-LY6G6F,anti-VSIG10, anti-TMEM25 and/or anti-LSR antibodies are used to maptheir epitopes on the LY6G6F, VSIG10, TMEM25 and/or LSR antigen,respectively. These different antibodies are coated on three differentsurfaces of the same chip to 8000 RUs each. Dilutions of each of themAbs are made, starting at 10 mu.g/mL and is incubated with Fc fusedLY6G6F, VSIG10, TMEM25 and/or LSR (50 nM) for one hour. The incubatedcomplex is injected over all the three surfaces (and a blank surface) atthe same time for 1.5 minutes at a flow rate of 20 .mu.L/min. Signalfrom each surface at end of 1.5 minutes, after subtraction ofappropriate blanks, has been plotted against concentration of mAb in thecomplex. Upon analysis of the data, the anti-LY6G6F, anti-VSIG10,anti-TMEM25 and/or anti-LSR antibodies are categorized into differentepitope groups depending on the epitope mapping results. The functionalproperties thereof are also compared.

Chinese hamster ovary (CHO) cell lines that express LY6G6F, VSIG10,TMEM25 and/or LSR protein at the cell surface are developed and used todetermine the specificity of the LY6G6F, VSIG10, TMEM25 and/or LSRHuMAbs by flow cytometry. CHO cells are transfected with expressionplasmids containing full length cDNA encoding a transmembrane forms ofLY6G6F, VSIG10, TMEM25 and/or LSR antigen or a variant thereof. Thetransfected proteins contained an epitope tag at the N-terminus are usedfor detection by an antibody specific for the epitope. Binding of aanti-LY6G6F, anti-VSIG10, anti-TMEM25 and/or anti-LSR MAb is assessed byincubating the transfected cells with each of the LY6G6F, VSIG10, TMEM25and/or LSR Abs at a concentration of 10 mu.g/ml. The cells are washedand binding is detected with a FITC-labeled anti-human IgG Ab. A murineanti-epitope tag Ab, followed by labeled anti-murine IgG, is used as thepositive control. Non-specific human and murine Abs are used as negativecontrols. The obtained data is used to assess the specificity of theHuMAbs for the LY6G6F, VSIG10, TMEM25 and/or LSR antigen target.

These antibodies and other antibodies specific to LY6G6F, VSIG10, TMEM25and/or LSR may be used in the afore-described anti-LY6G6F, anti-VSIG10,anti-TMEM25 and/or anti-LSR related therapies such as treatment ofcancers wherein LY6G6F, VSIG10, TMEM25 and/or LSR antigen isdifferentially expressed and/or for modulating (enhancing or inhibiting)B7 immune co-stimulation involving the LY6G6F, VSIG10, TMEM25 and/or LSRantigen such as in the treatment of cancers and autoimmune diseaseswherein such antibodies will e.g., prevent negative stimulation of Tcell activity against desired target cancer cells or prevent thepositive stimulation of T cell activity thereby eliciting a desiredanti-autoimmune effect.

The invention has been described and various embodiments providedrelating to manufacture and selection of desired anti-LY6G6F,anti-VSIG10, anti-TMEM25 and/or anti-LSR antibodies for use astherapeutics and diagnostic methods wherein the disease or condition isassociated with LY6G6F, VSIG10, TMEM25 and/or LSR antigen. Differentembodiments may optionally be combined herein in any suitable manner,beyond those explicit combinations and subcombinations shown herein. Theinvention is now further described by the claims which follow.

1-21. (canceled)
 22. A monoclonal or polyclonal antibody or an antigenbinding fragment thereof comprising an antigen binding site that bindsspecifically to an isolated polypeptide consisting essentially of anamino acid sequence as set forth in any one of SEQ ID NOs: 3-6, 60, 61,82-93 or 97-100.
 23. The antibody or the antigen binding fragment ofclaim 22, wherein the antigen binding site comprises a conformational orlinear epitope, and wherein the antigen binding site contains about 3-7contiguous or non-contiguous amino acids.
 24. The antibody or fragmentaccording to claim 23, wherein the antibody is a fully human antibody,chimeric antibody, humanized or primatized antibody.
 25. The antibody orthe antigen binding fragment according to claim 23, wherein the antibodyis selected from the group consisting of Fab, Fab′, F(ab′)2, F(ab′),F(ab), Fv or scFv fragment and minimal recognition unit.
 26. Theantibody or the antigen binding fragment according to claim 25, whereinthe antibody is coupled to a moiety selected from a drug, aradionuclide, a fluorophore, an enzyme, a toxin, a therapeutic agent, ora chemotherapeutic agent, and wherein the detectable marker is aradioisotope, a metal chelator, an enzyme, a fluorescent compound, abioluminescent compound or a chemiluminescent compound.
 27. The antibodyor the antigen binding fragment of claim 22, wherein said antibodyblocks or inhibits the interaction of a VSIG10 polypeptide with acounterpart, wherein said VSIG10 polypeptide is a polypeptide consistingessentially of the amino acid sequence set forth in any one of SEQ IDNOs: 3, 4, 5, 6, 60, 61; 82-93, or 97-100.
 28. (canceled)
 29. (canceled)30. (canceled)
 31. A pharmaceutical composition comprising an antibodyaccording to claim 22, and further comprising a pharmaceuticallyacceptable diluent or carrier.
 32. (canceled)
 33. A method for treatinga subject in need thereof for cancer, the method comprisingadministering to the subject an antibody according to claim
 22. 34.(canceled)
 35. A method of performing one or more of the following in asubject: a. upregulating cytokines; b. inducing expansion of T cells; c.promoting antigenic specific T cell immunity; d. promoting CD4+ and/orCD8+ T cell activation; comprising administering an antibody accordingto claim 22 to the subject.
 36. (canceled)
 37. (canceled)
 38. (canceled)39. (canceled)
 40. (canceled)
 41. (canceled)
 42. (canceled) 43.(canceled)
 44. (canceled)
 45. (canceled)
 46. (canceled)
 47. The methodof claim 33, wherein the treatment is combined with another moiety ortherapy useful for treating cancer.
 48. The method of claim 47, whereinthe therapy is radiation therapy, antibody therapy, chemotherapy,photodynamic therapy, adoptive T cell therapy, Treg depletion, surgeryor in combination therapy with conventional drugs.
 49. The method ofclaim 48, wherein the moiety is selected from the group consisting ofimmunosuppressants, cytotoxic drugs, tumor vaccines, antibodies (e.g.bevacizumab, erbitux), peptides, pepti-bodies, small molecules,chemotherapeutic agents such as cytotoxic and cytostatic agents (e.g.paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine,temozolomide, irinotecan, 5FU, carboplatin), immunological modifierssuch as interferons and interleukins, immunostimulatory antibodies,growth hormones or other cytokines, folic acid, vitamins, minerals,aromatase inhibitors, RNAi, Histone Deacetylase Inhibitors, andproteasome inhibitors.
 50. The method of claim 33 wherein the cancer isselected from a group consisting of breast cancer, cervical cancer,ovary cancer, endometrial cancer, melanoma, bladder cancer, lung cancer,pancreatic cancer, colon cancer, prostate cancer, leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma,Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin'slymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronicmyelogenous leukemia, thyroid cancer, thyroid follicular cancer,myelodysplastic syndrome (MDS), fibrosarcomas and rhabdomyosarcomas,melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma,glioblastoma, benign tumor of the skin, keratoacanthomas, renal cancer,anaplastic large-cell lymphoma, esophageal squamous cells carcinoma,hepatocellular carcinoma, follicular dendritic cell carcinoma,intestinal cancer, muscle-invasive cancer, seminal vesicle tumor,epidermal carcinoma, spleen cancer, bladder cancer, head and neckcancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancerof the retina, biliary cancer, small bowel cancer, salivary glandcancer, cancer of uterus, cancer of testicles, cancer of connectivetissue, prostatic hypertrophy, myelodysplasia, Waldenstrom'smacroglobinaemia, nasopharyngeal, neuroendocrine cancer, myelodysplasticsyndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid,oesophagogastric, fallopian tube cancer, peritoneal cancer, papillaryserous mullerian cancer, malignant ascites, gastrointestinal stromaltumor (GIST), Li-Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL),and wherein the cancer is non-metastatic, invasive or metastatic. 51.The method of claim 50, wherein the cancer is any of melanoma, cancer ofliver, renal, brain, breast, colon, lung, ovary, pancreas, prostate,stomach, multiple myeloma, Hodgkin's lymphoma, non Hodgkin's lymphoma,acute and chronic lymphoblastic leukemia and acute and chronic myeloidleukemia.
 52. A method for potentiating a secondary immune response toan antigen in a subject, which method comprises administering theantigen to the subject, wherein the antigen is a cancer antigen; andadministering effective amount of an antibody according to claim 22 tothe subject.
 53. (canceled)
 54. (canceled)
 55. A method of using anantibody according to claim 22 as a cancer vaccine adjuvant, comprisingadministration to a patient an immunogenic amount of a tumor associatedantigen preparation of interest; and a cancer vaccine adjuvant in aformulation suitable for immunization, wherein the immune responseagainst the tumor associated antigen in the presence of the cancervaccine adjuvant is stronger than in the absence of the cancer vaccineadjuvant. 56-69. (canceled)
 70. The method of claim 35, furthercomprising treating cancer through administration of said antibody tothe subject.